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The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli . Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto- N -tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.

Advancing the formation of artificial membraneless compartments with organizational complexity and diverse functionality remains a challenge. Typically, synthetic compartments or membraneless organelles are made up of intrinsically disordered proteins featuring low-complexity sequences or polypeptides with repeated distinctive short linear motifs. In order to expand the repertoire of tools available for the formation of synthetic membraneless compartments, here, a range of DIshevelled and aXin (DIX) or DIX-like domains undergoing head-to-tail polymerization were demonstrated to self-assemble into aggregates and generate synthetic compartments within E. coli cells. Then, synthetic complex compartments with diverse intracellular morphologies were generated by coexpressing different DIX domains. Further, we genetically incorporated a pair of interacting motifs, comprising a homo-dimeric domain and its anchoring peptide, into the DIX domain and cargo proteins, respectively, resulting in the alteration of both material properties and client recruitment of synthetic compartments. As a proof-of-concept, several human milk oligosaccharide biosynthesis pathways were chosen as model systems. The findings indicated that the recruitment of pathway sequential enzymes into synthetic compartments formed by DIX-DIX heterotypic interactions or by DIX domains embedded with specific interacting motifs efficiently boosted metabolic pathway flux and improved the production of desired chemicals. We propose that these synthetic compartment systems present a potent and adaptable toolkit for controlling metabolic flux and facilitating cellular engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The regulator of capsule synthesis (Rcs) system, an atypical two-component system prevalent in numerous gram-negative bacteria, serves as a sophisticated regulatory phosphorylation cascade mechanism. It plays a pivotal role in perceiving environmental stress and regulating the expression of downstream genes to ensure host survival. During the signaling transduction process, various proteins participate in phosphorylation to further modulate signal inputs and outputs. Although the structure of core proteins related to the Rcs system has been partially well-defined, and two models have been proposed to elucidate the intricate molecular mechanisms underlying signal sensing, a systematic characterization of the signal transduction process of the Rcs system remains challenging. Furthermore, exploring its corresponding regulator outputs is also unremitting. This review aimed to shed light on the regulation of bacterial virulence by the Rcs system. Moreover, with the assistance of the Rcs system, biosynthesis technology has developed high-value target production. Additionally, via this review, we propose designing chimeric Rcs biosensor systems to expand their application as synthesis tools. Finally, unsolved challenges are highlighted to provide the basic direction for future development of the Rcs system., Competing Interests: Declaration of Competing Interest The authors have read the journal’s policy and have no conflicts., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Lacto- N -neotetraose (LNnT), a prominent neutral human milk oligosaccharide (HMO), serves as a pivotal structural element in complex HMO biosynthesis. Given its promising health effects for infants, the biosynthesis of LNnT is garnering greater interest. Using a previously engineered strain as a chassis, a highly effective LNnT producer was constructed. First, LNnT synthesis in Escherichia coli MG1655 was achieved by introducing β1,3- N -acetylglucosaminyltransferase LgtA and β1,4-galactosyltransferase CpsIaJ, coupled with the optimization of enzyme expression levels using various promoters. Subsequently, ugd underwent disruption, and the galE gene was enhanced by replacing its promoter with P J23119 or P tac . Then, a lux-type quorum sensing (QS) system was applied to achieve varied metabolic regulation. Additionally, systematic optimization of the QS promoters was conducted to further improve the LNnT titer in the shake flask. Finally, the extracellular titer of LNnT was 20.33 g/L, accompanied by a productivity of 0.41 g/L/h.

Lacto- N -biose I (LNB), termed a Type 1 disaccharide, is an important building block of human milk oligosaccharides. It shows promising prebiotic activity by stimulating the proliferation of many gut-associated bifidobacteria and thus displays good potential in infant foods or supplements. Enzymatic and microbial approaches to LNB synthesis have been studied, almost all of which involve glycosylation of LNB phosphorylase as the final step. Herein, we report a new and easier microbial LNB synthesis strategy through the route "lactose → lacto- N -triose II (LNTri II) → lacto- N -tetraose (LNT) → LNB". A previously constructed LNT-producing Escherichia coli BL21(DE3) strain was engineered for LNB biosynthesis by introducing Bifidobacterium bifidum LnbB. LNB was efficiently produced, accompanied by lactose regeneration. Genomic integration of key pathway genes related to LNTri II and LNT synthesis was performed to enhance LNB titers. The final engineered strain produced 3.54 and 26.88 g/L LNB by shake-flask and fed-batch cultivation, respectively.

Lacto-N-fucopentaose I (LNFP I) is an abundant and important fucosylated human milk oligosaccharide (HMO). Here, an efficient LNFP I-producing strain without by-product 2'-fucosyllactose (2'-FL) was developed by advisable stepwise de novo pathway construction in Escherichia coli. Specifically, the genetically stable lacto-N-triose II (LNTri II)-producing strains were constructed by the multicopy integration of β1,3-N-acetylglucosaminyltransferase. LNTri II can be further converted to lacto-N-tetraose (LNT) by LNT-producing β1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific α1,2-fucosyltransferase was verified to eliminate by-product 2'-FL, and binding free energy of the complex was analyzed to explain the product distribution. Subsequently, further attempts aiming to improve α1,2-fucosyltransferase activity and the supply of GDP-fucose were carried out. Our engineering strategies enabled the stepwise de novo construction of strains that produced up to 30.47 g/L of extracellular LNFP I, without accumulation of 2'-FL, and with only minor intermediates residue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lacto- N -tetraose (LNT) is an important neutral human milk oligosaccharide (HMO) and acts as a significant core structure for complex HMO biosynthesis. We previously achieved high-yield LNT biosynthesis (57.5 g/L) using fed-batch fermentation; however, residual lacto- N -triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNT - producing Escherichia coli with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of lgtA (encoding β1,3- N -acetylglucosaminyltransferase). Comparable and low titers of LNT (3.73-4.61 g/L) and LNTri II (0.33-0.63 g/L), respectively, were obtained by introducing β1,3-galactosyltransferase. To reduce residual LNTri II, the E. coli transporter gene setA was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding β- N -acetylhexosaminidase ( BbhI ) was introduced into LNT-producing strains or E. coli BL21(DE3) for single- or mixed-strain cultivation, respectively. Finally, LNT was obtained (30.13 g/L) in a cocultivation system of mixed engineered strains without undesired LNTri II.

Human milk oligosaccharides are complex, indigestible oligosaccharides that provide ideal nutrition for infant development. Here, 2'-fucosyllactose was efficiently produced in Escherichia coli by using a biosynthetic pathway. For this, both lacZ and wcaJ (encoding β-galactosidase and UDP-glucose lipid carrier transferase, respectively) were deleted to enhance the 2'-fucosyllactose biosynthesis. To further enhance 2'-fucosyllactose production, SAMT from Azospirillum lipoferum was inserted into the chromosome of the engineered strain, and the native promoter was replaced with a strong constitutive promoter (P J23119 ). The titer of 2'-fucosyllactose was increased to 8.03 g/L by introducing the regulators rcsA and rcsB into the recombinant strains. Compared to wbgL-based strains, only 2'-fucosyllactose was produced in SAMT-based strains without other by-products. Finally, the highest titer of 2'-fucosyllactose reached 112.56 g/L in a 5 L bioreactor by fed-batch cultivation, with a productivity of 1.10 g/L/h and a yield of 0.98 mol/mol lactose, indicating a strong potential in industrial production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI , fucK , and rhaA . The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

As one of the most abundant components in human milk oligosaccharides, 2'-fucosyllactose (2'-FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine-tuning the expression of pathway enzymes and achieved a striking increase of 2'-FL production, directly facilitating the metabolic flux toward the key intermediate GDP-l-fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2'-FL. Specifically, we first fine-tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2'-FL synthesis. Then, two key enzymes (GDP-mannose 4,6-dehydratase and GDP- l-fucose synthase) were self-assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD-RIDD (RI anchoring disruptor-RI dimer D/D domains), resulting in noticeable improvement of 2'-FL production. Next, to further strengthen the metabolic flux toward 2'-FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher-SpyTag or PDZ-PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2'-FL biosynthesis and showed a 2.1-fold increase of 2'-FL production compared with a strain expressing free-floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2'-FL in shake flask fermentation and was capable of producing 25.1 g/L 2'-FL by fed-batch cultivation. This work provides novel approaches for further improvement and large-scale production of 2'-FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain., (© 2022 Wiley Periodicals LLC.)

3'-Sialyllactose (3'-SL) is one of the most important and simplest sialylated human milk oligosaccharides. In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was applied for 3'-SL production. Specifically, the precursor CMP-Neu5Ac synthesis pathway genes and α2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21(DE3)Δ lacZ to realize 3'-SL synthesis. Genes nanA and nanK involved in Neu5Ac catabolism were further deleted to reduce the metabolic flux of competitive pathway. Several α2,3-sialyltransferases from different species were selected to evaluate the sialylation effect. The precursor pools were balanced and improved by optimizing key enzyme expression involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathway. Finally, an additional α2,3-sialyltransferase expression cassette was integrated into chromosome to maximize 3'-SL synthesis, and 4.5 g/L extracellular 3'-SL was produced at a shake-flask level. The extracellular 3'-SL concentration was raised to 23.1 g/L in a 5 L bioreactor fermentation, which represents the highest extracellular value ever reported.

Recently, the biosynthesis of human milk oligosaccharides (HMOs) has been attracting increasing attention. Lacto- N -neotetraose (LNnT) is one of the most important neutral-core HMOs with promising health effects for infants. It has received Generally Recognized as Safe (GRAS) status and is the second HMO commercially added in infant formula after 2'-fucosyllactose. In previous studies, a series of engineered Escherichia coli strains have been constructed and optimized to produce high titers of precursor lacto- N -triose II. On the basis of these strains, LNnT-producing strains were constructed by overexpressing the β1,4-galactosyltransferase-encoding gene from Aggregatibacter actinomycetemcomitans NUM4039 (Aa - β1,4 - GalT). Interestingly, an appreciable LNnT titer was obtained by weakening the metabolic flux of the UDP-GlcNAc pathway and simply overexpressing the essential genes lgtA, galE, and Aa - β1 , 4 - GalT in lacZ-, wecB - , and nagB-deleted E. coli . Subsequently, LNnT synthesis was optimized through balancing the expression of these three biosynthetic enzymes. The optimized strain produced LNnT with an extracellular titer of 12.1 g/L in fed-batch cultivation, with the productivity and specific yield of 0.25 g/L·h and 0.27 g/g dry cell weight, respectively.

2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk. In this study, a highly efficient biosynthetic route for 2'-FL production was designed via the de novo pathway of GDP-l-fucose using engineered Escherichia coli BL21(DE3). Specifically, plasmid-based strains with previously deleted lacZ and wcaJ were further reconstructed by introducing de novo pathway genes and α1,2-fucosyltransferase-encoding wbgL to realize 2'-FL synthesis. The 2'-FL titer was enhanced to 3.92 g/L by further introducing rcsA and rcsB . Subsequently, the additional wbgL expression cassette was chromosomally integrated into recA locus to strengthen fucosylation reaction and a strong constitutive promoter (P J23119 ) was used to replace the original promoters of manC-manB and gmd-wcaG to improve 2'-FL synthesis. The maximal 2'-FL titer reached 9.06 and 79.23 g/L in shake-flask and fed-batch cultivation, respectively. The 2'-FL productivity reached 1.45 g/L/h, showing remarkable production potential in large-scale industrial application.

Recently, human milk oligosaccharides (HMOs) have attracted increasing attention and display great commercial importance, especially for the infant formula industry. Lacto- N -tetraose (LNT) is an important neutral HMO commercially added in infant formula and a core structure for synthesizing complex HMOs. Previously, a novel LNT-generating β-1,3-galactosyltransferase from Pseudogulbenkiania ferrooxidans was identified and used for construction of an LNT-producing engineered Escherichia coli . In this work, LNT biosynthesis was further enhanced by pathway optimization and uridine 5'-triphosphate (UTP) regeneration. The main strategies included genomic integration of UDP-glucose 4-epimerase-encoding gene, fine-tuning of the LNT pathway-related genes, blocking of competitive pathways related to UDP-galactose, and overexpression of UTP supply related genes. The maximal LNT titer reached 6.16 and 57.5 g/L by shake-flask and fed-batch fermentation, respectively.

As one of the attractive fucosylated human milk oligosaccharides, the biological production of 3-fucosyllactose (3-FL) has received great attention, as it exhibits many excellent physiological functions for infants. In this work, a novel 3-FL-producing α-1,3-fucosyltransferase (α1,3-FucT) named FutM2 from Bacteroides gallinaceum was first selected from nine potential candidates in the NCBI database. Then, a highly 3-FL-producing engineered Escherichia coli strain was constructed by modular pathway enhancement including the GDL-l-fucose precursor supply by overexpressing manC, manB, gmd, and wcaG (CBGW), and the 3-FL synthesis pathway by introducing B. gallinaceum FutM2. Finally, a titer of 20.3 g L -1 and productivity of 0.40 g L -1 h -1 of 3-FL were achieved in the 3-L bioreactor by engineered E. coli (Δ lacZ Δ wcaJ ) harboring pCDF- CBGW and pET- futM2 . Our study provided a novel α1,3-FucT from B. gallinaceum that could be used for 3-FL production, presenting an efficient microbial cell factory platform to de novo synthesize 3-FL from glycerol.

2'-Fucosyllactose (2'-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2'-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2'-FL biosynthesis in recombinant Escherichia coli . First, the salvage pathway for 2'-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E. coli BL21(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2'-FL biosynthesis. Moreover, to further improve the yield and titer of 2'-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo . The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2'-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2'-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2'-FL biosynthesis in engineered E. coli .

Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto- N -tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing β-1,3-galactosyltransferase (β-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto- N -triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans β-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L·h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

L-Asparaginase (L-ASNase, EC 3.5.1.1), an antitumor drug for acute lymphoblastic leukemia (ALL) therapy, is widely used in the clinical field. Similarly, L-ASNase is also a powerful and significant biological tool in the food industry to inhibit acrylamide (AA) formation. This review comprehensively summarizes the latest achievements and improvements in the production, modification, and application of microbial L-ASNase. To date, the expression levels and optimization of expression hosts such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, have made significant progress. In addition, examples of successful modification of L-ASNase such as decreasing glutaminase activity, increasing the in vivo stability, and enhancing thermostability have been presented. Impressively, the application of L-ASNase as a food addition aid, as well as its commercialization in the pharmaceutical field, and cutting-edge biosensor application developments have been summarized. The presented results and proposed ideas could be a good guide for other L-ASNase researchers in both scientific and practical fields., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lacto- N -triose II (LNT II), a core structural unit of human milk oligosaccharides (HMOs), has attracted substantial attention for its nutraceutical potentials and applications in the production of complex HMOs. In this study, Escherichia coli was metabolically engineered to efficiently produce LNT II using glycerol as a carbon source and lactose as a substrate. The UDP- N -acetylglucosamine (UDP-GlcNAc) biosynthesis pathway was strengthened, and β-1,3- N -acetylglucosaminyltransferase (LgtA) was introduced to construct an LNT II-producing base strain. To increase the titer and yield of LNT II, combinatorial optimization of the copy number and the ribosomal binding site sequence was performed to tune the gene expression strength and translation rates of the pathway enzymes. Next, multipoint mutations were introduced to glucosamine-6-phosphatesynthase (GlmS) to relieve the feedback inhibition. Then, a series of engineered strains were constructed by blocking the futile pathways by the deletion of the relevant genes. Finally, the culture conditions were optimized. LNT II titer was improved step-by-step from 0.53 to 5.52 g/L in shake-flask cultivations. Fed-batch culture of the final engineered strain produced 46.2 g/L of LNT II, with an LNT II productivity and content of 0.77 g/(L·h) and 0.95 g/g dry cell weight, respectively.

Guanosine 5'-diphosphate (GDP)-l-fucose is an important nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Here, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP-l-fucose in engineered Escherichia coli . The de novo GDP-l-fucose synthesis pathway was partitioned into two modules and fine-tuned at both transcriptional and translational levels, which remarkably improved the GDP-l-fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP-l-fucose to colanic acid. Furthermore, cofactors were regenerated to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve a titer of 18.33 mg/L in shake-flask cultivation, showed 106.21 mg/L intracellular GDP-l-fucose accumulation and a DCW-specific GDP-l-fucose content of 4.28 mg/g through fed-batch cultivation. In general, this study demonstrated that the utilization of combinatorial modular pathway engineering significantly improved the de novo synthesis of GDP-l-fucose in engineered E. coli .

Background: d-Mannose is not only the epimer of d-glucose at the C-2 position, but also the aldose isomer of d-fructose. Because of its physiological properties and health benefits, d-mannose has attracted public interest. It has been confirmed that d-mannose has broad applications in food, cosmetics, and pharmaceutical industries. According to the Izumoring strategy, d-glucose isomerase (d-GI) and d-lyxose isomerase (d-LI) play important roles in the conversions of d-fructose from d-glucose and of d-mannose from d-fructose respectively. In this study, a one-step enzyme process of d-mannose production from d-glucose has been constructed by co-expression of the d-GI from Acidothermus cellulolyticus and d-LI from Thermosediminibacter oceani in Escherichia coli BL21(DE3) cells., Results: The co-expression system exhibits maximum activity at pH 6.5 and 65 °C with Co 2+ supplement. It is relatively thermostable at less than 65 °C. When the reaction reaches equilibrium, the ratio of d-glucose, d-fructose, and d-mannose is approximately 34 : 49.6 : 16.4. By using this co-expression system, about 60.0 g L -1 d-mannose is obtained from 400 g L -1 d-glucose in 8 h., Conclusion: This co-expression of d-GI and d-LI system provides a novel and efficient approach for d-mannose production. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG., Results: The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co 2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively., Conclusion: A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L -1 d-glucose as a substrate, 204.3 g L -1 d-fructose and 89.1 g L -1 d-allulose were produced. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

This paper presents results on immunobeads-based isolation of rare bacteria and their capture at a boron-doped ultrananocrystalline diamond (BD-UNCD) electrode in a microfluidic dielectrophoretic preconcentrator. We systematically vary the bead surface chemistry and the BD-UNCD surface chemistry and apply dielectrophoresis to improve the specific and the nonspecific capture of bacteria or beads. Immunobeads were synthesized by conjugating antibodies to epoxy-/sulfate, aldehyde-/sulfate, or carboxylate-modified beads with or without poly(ethylene glycol) (PEG) coimmobilization. The carboxylate-modified beads with PEG provided the highest capture efficiency (∼65%) and selectivity (∼95%) in isolating live Escherichia coli O157:H7 from cultures containing 1000 E. coli O157:H7 colony-forming units (cfu)/mL, or ∼500 E. coli O157:H7 and ∼500 E. coli K12 cfu/mL. Higher specificity was achieved with the addition of PEG to the antibody-functionalized bead surface, highest with epoxy-/sulfate beads (85-86%), followed by carboxylate-modified beads (76-78%) and aldehyde-/sulfate beads (74-76%). The bare BD-UNCD electrodes of the preconcentrator successfully withstood 240 kV/m for 100 min that was required for the microfluidic dielectrophoresis of 1 mL of sample. As expected, the application of dielectrophoresis increased the specific and the nonspecific capture of immunobeads at the BD-UNCD electrodes; however, the capture specificity remained unaltered. The addition of PEG to the antibody-functionalized BD-UNCD surface had little effect on the specificity in immunobeads capture. These results warrant the fabrication of electrical biosensors with BD-UNCD so that dielectrophoretic preconcentration can be performed directly at the biosensing electrodes.

Resistance to β-lactam antibiotics through β-lactamase production by Enterobacteriaceae continues to burden the health-care sector worldwide. Traditional methods for detection of β-lactamases are time-consuming and labor-intensive and newer methods with varying capabilities continue to be developed. The objective of this study was to develop a multiplex PCR (M-PCR) system for the detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes and to apply it in clinical Klebsiella pneumoniae and Escherichia coli strains. To do this, we used group-specific PCR primers in singleplex reactions followed by optimization into multiplex reactions. Specificity and sensitivity of the M-PCR were then evaluated using 58 reference strains before its application to detect bla group genes in 203 clinical Enterobacteriaceae strains. PCR amplicons were sequenced to determine the β-lactamase subtypes. The M-PCR system exhibited 100% specificity and sensitivity. In all, 83.7% of K. pneumoniae and 89.8% of E. coli clinical strains harbored bla group genes with 46.9%, 40.1%, 15.0%, 21.1% and 6.1% of K. pneumoniae having blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively, whereas 12.2%, 77.6%, 22.4%, 36.7% and 8.2% of E. coli had blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively. BlaSHV-1, blaSHV-11, blaSHV-27, blaSHV-33, blaSHV-144, blaTEM-1, blaTEM-135, blaOXA-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-65 and blaCTX-M-104 were detected. In conclusion, the M-PCR system was efficient and versatile with an advantage of simultaneously detecting all the targeted bla group genes. Hence, it is a potential candidate for developing M-PCR kits for the screening of these genes for clinical or epidemiological purposes.

The objective of this study was to compare quinolone resistance and gyrA mutations in clinical isolates of Klebsiella pneumoniae and Escherichia coli from Chinese adults who used quinolone in the preceding month and children without any known history of quinolone administration. The antimicrobial susceptibilities of 61 isolates from children and 79 isolates from adults were determined. The mutations in the quinolone resistance-determining regions in gyrA gene were detected by PCR and DNA sequencing. Fluoroquinolone resistance and types of gyrA mutations in isolates from children and adults were compared and statistically analyzed. No significant differences were detected in the resistance rates of ciprofloxacin and levofloxacin between children and adults among isolates of the two species (all P > 0.05). The double mutation Ser83→Leu + Asp87→Asn in the ciprofloxacin-resistant isolates occurred in 73.7% isolates from the children and 67.9% from the adults, respectively (P = 0.5444). Children with no known history of quinolone administration were found to carry fluoroquinolone-resistant Enterobacteriaceae isolates. The occurrence of ciprofloxacin resistance and the major types of gyrA mutations in the isolates from the children were similar to those from adults. The results indicate that precautions should be taken on environmental issues resulting from widespread transmission of quinolone resistance.

Background: Wide use of ciprofloxacin and levofloxacin has often led to increased resistance. The resistance rate to these two agents varies in different clinical isolates of Enterobacteriaceae. Mutations of GyrA within the quinolone resistance-determining regions have been found to be the main mechanism for quinolone resistance in Enterobacteriaceae. It has been shown that only some of the mutations in the gyrA gene identified from clinical sources were involved in fluoroquinolone resistance. Whether different patterns of gyrA mutation are related to antimicrobial resistance against ciprofloxacin and levofloxacin is unclear., Methods: The minimum inhibitory concentration (MIC) of ciprofloxacin and levofloxacin were determined by the agar dilution method followed by PCR amplification and sequencing of the quinolone resistance determining region of gyrA to identify all the mutation types. The correlation between fluoroquinolone resistance and the individual mutation type was analyzed., Results: Resistance differences between ciprofloxacin and levofloxacin were found in 327 isolates of K. pneumoniae and E. coli in Harbin, China and in the isolates reported in PubMed publications. GyrA mutations were found in both susceptible and resistant isolates. For the isolates with QRDR mutations, the resistance rates to ciprofloxacin and levofloxacin were also statistically different. Among the 14 patterns of alterations, two single mutations (Ser83Tyr and Ser83Ile), and three double mutations (Ser83Leu+Asp87Asn, Ser83Leu+Asp87Tyr and Ser83Phe+Asp87Asn) were associated with both ciprofloxacin and levofloxacin resistance. Two single mutations (Ser83Phe and Ser83Leu) were related with ciprofloxacin resistance but not to levofloxacin. Resistance difference between ciprofloxacin and levofloxacin in isolates harboring mutation Ser83Leu+Asp87Asn were of statistical significance among all Enterobacteriaceae (P<0.001)., Conclusions: Resistance rate to ciprofloxacin and levofloxacin were statistically different among clinical isolates of Enterobacteriaceae harboring GyrA mutations. Ser83Leu+Asp87Asn may account for the antimicrobial resistance difference between ciprofloxacin and levofloxacin.

l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout sucAB and aceAK gene in the TCA cycle pathway of Escherichia coli (E. coli). For single-gene knockout, the whole process took approximately 7 days. However, the manipulation time was reduced by 2 days for each round of gene modification for multigene editing. Using the genome-edited recombinant strain E. coli BL21(DE3) ΔsucABΔaceAK/pET-28a(+)-ido (2Δ-ido), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to E. coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production. [ABSTRACT FROM AUTHOR]

• A novel D-LI was characterized from Thermoflavimicrobium dichotomicum. • It was strictly metal-dependent and showed maximum activity at 60 °C and pH 7.5. • Equilibrium ratio of d -mannose and d -fructose was measured to be 25:75. • From 500 g/L of d -fructose, 110.5 g/L of d -mannose was produced by T. dichotomicum D-LI. d -Mannose is the aldose isomer of d -fructose and displays unique physiological functions and health applications. As a result, it has attracted increasing interest from the public. Because of its wide substrate specificity, d -lyxose isomerase (D-LI) has been applied to d -mannose bioproduction. In this article, the Thermoflavimicrobium dichotomicum D-LI encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). The novel protein T. dichotomicum D-LI was identified and characterized, and its maximum enzyme activity was exhibited at pH 7.5 (phosphate buffer) and 60 °C in the presence of Mn2+. Similar to other D-LIs, T. dichotomicum D-LI formed a homodimeric structure and showed strict conservation of the metal coordination and substrate binding sites. The D-LI half-life (t 1/2) was 9.91 h and 3.05 h at 55 and 60 °C, respectively, and its melting temperature (T m) was 72.85 °C. T. dichotomicum D-LI displayed the highest specific activity towards d -lyxose among the substrates tested, followed by d -mannose. It also efficiently converted d -fructose to d -mannose, and the equilibrium ratio between d -mannose and d -fructose during T. dichotomicum D-LI activity was 25:75. From 500 g/L d -fructose, 110.5 g/L d -mannose was produced after 6 h, suggesting that T. dichotomicum D-LI might provide an alternative for d -mannose production. [ABSTRACT FROM AUTHOR]

Highlights • An endoinulinase was characterized from Aspergillus arachidicola. • The optimum pH and temperature were pH 5.5 and 60 °C. • DP3 and DP4 were produced as major products during inulin endohydrolysis. • The conversion yield of inulin to inulooligosaccharides reached 60–90%. • Yeast treatment was first developed to improve the inulooligosaccharides purity. Abstract Inulin has been widely used as a cheap material to produce many valuable products such as inulooligosaccharides. In this study, an inulooligosaccharide-producing endoinulinase was identified from Aspergillus arachidicola. The gene encoding the mature endoinulinase was cloned and intracellularly overexpressed in Escherichia coli. The recombinant endoinulinase was purified by nickel affinity chromatography. The optimum pH and temperature were pH 5.5 and 60 °C, respectively. It was highly thermostable at up to 55 °C for 2 h, however easily inactivated at higher temperatures. The enzyme efficiently produced inulooligosaccharides from inulin, with inulotriose and inulotetraose as major products. When 10, 30, 50, and 100 g L−1 of inulin was used as substrate, the final conversion ratio of total inulooligosaccharides reached 88.4%, 83.5%, 80.2%, and 60.1%, respectively. In addition, yeast treatment was first developed to improve the inulooligosaccharides purity by specifically consuming sucrose and monosaccharides without any losses in inulooligosaccharides content. [ABSTRACT FROM AUTHOR]

The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Inevitably, considering its extensive use and misuse, resistance toward ciprofloxacin has increased in almost all clinically relevant bacteria. This study aimed to investigate the transcriptome changes at a high concentration of ciprofloxacin in Escherichia coli. In brief, 1,418 differentially expressed genes (DEGs) were identified, from which 773 genes were upregulated by ciprofloxacin, whereas 651 genes were downregulated. Enriched biological pathways reflected the upregulation of biological processes such as DNA damage and repair system, toxin/antitoxin systems, formaldehyde detoxification system. With kyoto encyclopedia of genes and genomes pathway analysis, higher expressed DEGs were associated with "LPS biosynthesis," "streptomycin biosynthesis," and "polyketide sugar unit biosynthesis." Lower expressed DEGs were associated with "biosynthesis of amino acids" and "flagellar assembly" pathways. After treatment of ciprofloxacin, lipopolysaccharide (LPS) release was increased by two times, and the gene expression level of LPS synthesis was elevated (p < 0.05) in both reference and clinical strains. Our results demonstrated that transient exposure to high-dose ciprofloxacin is a double-edged sword. Cautions should be taken when administering high-dose antibiotic treatment for infectious diseases. [ABSTRACT FROM AUTHOR]

• A novel methyl parathion hydrolase was investigated. • Leveraging 1 mM Mn2+, the enzymatic activity would be significantly enhanced. • Co-incubation of 1 mM Mn2+ would greatly increase the T m of free-enzyme. • The MPH showed high effectiveness in the degradation of various OPs pesticides. Organophosphorus pesticides are highly toxic phosphate compounds with the general structure of O = P(OR) 3 and threaten human health seriously. Methyl parathion hydrolase from microbial is an important enzyme to degrade organophosphorus pesticides (OPs) into less toxic or nontoxic compounds like. p-nitrophenol and diethyl phosphate. Here, a gene encoding methyl parathion hydrolase from Azohydromonas australica was firstly cloned and expressed in Escherichia coli. The recombinant hydrolase showed its optimal pH and temperature at pH 9.5 and 50 °C. Leveraging 1 mM Mn2+, the enzyme activity was significantly enhanced by 29.3-fold, and the thermostability at 40 and 50 °C was also improved. The recombinant MPH showed the specific activity of 4.94 and 16.0 U/mg towards methyl parathion and paraoxon, respectively. Moreover, A. australica MPH could effectively degrade various of OPs pesticides including methyl parathion, paraoxon, dichlorvos and chlorpyrifos in a few minutes, suggesting a great potential in the bioremediation of OPs pesticides. [ABSTRACT FROM AUTHOR]

• An exoinulinase was identified from a novel strain, A. piperis CBS 112811. • The enzyme displayed the maximal activity as 3750 U mg−1 at pH 6.0 and 55 °C. • The enzyme was stable up to 60 °C for 5 h, showing a relatively high thermostability. • One-pot production of d -allulose was developed using exoinulinase and DAEase. • The yield of d -allulose reached 21.4 g L−1 with 100 g L−1 inulin as a substrate using the two-enzyme system. Inulin has been widely used as a cheap bioresource for producing many valuable products by enzymatic hydrolysis or microbial fermentation, such as high-fructose syrup and fructooligosaccharides. In this work, a one-pot two-enzyme reaction system was developed to produce d -allulose from inulin using A. piperis exoinulinase and Dorea sp. d -Allulose 3-epimerase. The exoinulinase that was identified from Aspergillus piperis CBS 112811 was cloned and intracellularly expressed in Escherichia coli. The enzyme displayed the maximal activity as 3750 U mg−1 at pH 6.0 and 55 °C. For the effects of different cations, Mn2+ simulated the enzyme activity by 41 %. When 10 g L−1 inulin was hydrolyzed by A. piperis exoinulinase, the conversion rate reached 98 % within 6 h. Furthermore, the optimum pH, temperature and the ratio of the two enzymes loaded for one-pot reaction were measured to be pH 6.0, 60 °C and 15/15 U mL−1, respectively. The conversion rate of inulin to d -allulose reached 23.3 % after reaction for 4 h with 10 g L−1 inulin. When adding 100 g L−1 as a substrate, 21.4 g L−1 d -allulose was produced using the two-enzyme system. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A recombinant IFTase was identified from Arthrobacter chlorophenolicus A6. • The highest activity (902 U mg−1) was shown at 65 °C and pH 5.5. • It showed remarkable thermostability and was stable at up to 80 °C for 1 h. • The conversion ratio of inulin to DFA III was 81% when 50 g l−1 inulin was used. Abstract The gene encoding difructose anhydride III (DFA III)-forming inulin fructotransferase (IFTase) from Arthrobacter chlorophenolicus A6 was cloned and overexpressed in Escherichia.coli. The recombinant IFTase (DFA III-forming) was purified using one-step nickel affinity chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses, the enzyme showed a homotrimeric form composed of three identical subunits, each with an apparent molecular mass of 43 kDa. The maximum catalytic activity was shown at 65 °C and pH 5.5, and the specific activity was measured as 902 U mg−1. The enzyme showed remarkable thermostability and over half of the initial activity was retained after incubation at 80 °C for 1 h. The K m and V max were estimated to 12.93 mM and 2.89 μmol min−1 ml−1, respectively. After complete hydrolysis of inulin for DFA III production by the purified enzyme, the produced minor products contained sucrose (GF), 1-kestose (GF 2), and nystose (GF 3). The smallest substrate was identified to be GF 3. When 50, 100, and 200 g l−1 of inulin were hydrolyzed by the purified IFTase, the DFA III yield reached 81%, 72%, and 67%, respectively. [ABSTRACT FROM AUTHOR]

Currently, people pay more attention to marine sugars, because of their unique physiological effects. Alginate oligosaccharides (AOS) are the degradation products of alginate and have been used in food, cosmetic, and medicine fields. AOS display good physical characteristics (low relative molecular weight, good solubility, high safety, and high stability) and excellent physiological functions (immunomodulatory, antioxidant, antidiabetic, and prebiotic activities). Alginate lyase plays a key role in the AOS bioproduction. In this study, a novel PL-31 family alginate lyase from Paenibacillus ehimensis (paeh-aly) was identified and characterized. It was extracellularly secreted in E. coli and exhibited a preference for the substrate poly β - D -mannuronate. Using sodium alginate as the substrate, it showed the maximum catalytic activity (125.7 U/mg) at pH 7.5 and 55 °C with 50 mM NaCl. Compared with other alginate lyases, paeh-aly exhibited good stability. About 86.6% and 61.0% residual activity could be maintained after 5 h incubation at 50 and 55 °C respectively, and its T m value was 61.5 °C. The degradation products were AOS with DP 2–4. Paeh-aly demonstrated strong promise for AOS industrial production because of its excellent thermostability and efficiency. • A novel alginate lyase from Paenibacillus ehimensis (paeh-aly) was identified and characterized. • Paeh-aly belongs to PL-31 family, and shows poly β - D -mannuronate preference. • Paeh-aly exhibites maximum activity at 55 °C and pH 7.5. • Compared with other alginate lyases, paeh-aly exhibites good stability. • The degradation products are alginate oligosaccharides with DP 2–4. [ABSTRACT FROM AUTHOR]

• A novel d -LIase from C. polysaccharolyticus was biochemically characterized. • The recombinant d -LIase exhibited maximum activity at 65 °C and pH 6.5. • The d -LIase shows a homodimeric structure and quite thermostable up to 60 °C. • A high conversion (25.6 %) was achieved for d -mannose production using the d -LIase. Recently, functional sugars, such as d -mannose, have attracted considerable attention due to their excellent physiological benefits for human health and wide applications in food and pharmaceutical industries. Therefore, d -mannose production using a sugar isomerase such as d -lyxose isomerase (d -LIase) has emerged as a research hotspot owing to its advantages over plant extraction and chemical synthesis methods. In this study, a putative d -LIase gene from Caldanaerobius polysaccharolyticus was cloned and expressed in Escherichia coli. Then, a biochemical characterization of the recombinant d -LIase was carried out and its potential use in d -mannose production also assessed. Results showed that d -LIase exhibited its maximum activity under these optimal conditions: temperature of 65 °C, a pH of 6.5, and the Mn2+ metal ion. The d -LIase was active at pH 6.0–8.0; it was also quite thermostable up to 60 °C and approximately 85 % of its maximum activity was retained after incubating for 4 h. Further, our Nano-DSC analysis determined that its melting temperature (T m) was 70.74 °C. Using 100, 300, and 500 g L−1 of d -fructose as substrate, 25.6, 74.4, and 115 g L−1 of d -mannose were produced respectively, corresponding to a conversion rate of 25.6 %, 24.8 %, and 23.0 % under optimal conditions. Taken together, our results provide evidence for a promising candidate d -LIase for producing d -mannose directly from d -fructose. [ABSTRACT FROM AUTHOR]

• l -Arabinose isomerase and d -lyxose isomerase were co-expressed in E. coli BL21(DE3). • The co-expressed system exhibited optimal activity at 70 °C, pH 6.0. • l -Ribose was produced using recombinant E. coli cells through a one-pot process. • 20.9 g/L of l -ribose was produced from 100 g/L of l -arabinose using recombinant E. coli cells. l- Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l- ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l- ribose from the inexpensive starting material l- arabinose. The l- arabinose isomerase (l- AIase) gene from Alicyclobacillus hesperidum and the d- lyxose isomerase (d- LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli , resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L−1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L−1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L−1 of l- ribose from initial l- arabinose concentrations of 100, 300, and 500 g L−1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l- ribose from l- arabinose under slightly acidic conditions using a co-expression system harboring l- AIase and d- LIase genes. [ABSTRACT FROM AUTHOR]