lacZ Research Articles

Engineering Escherichia coli MG1655 for Highly Efficient Biosynthesis of 2'-Fucosyllactose by De Novo GDP-Fucose Pathway.

2'-Fucosyllactose (2'-FL), the most typical human milk oligosaccharide, is used as an additive in premium infant formula. Herein, we constructed two highly effective 2'-FL synthesis producers via a de novo GDP-fucose pathway from engineered Escherichia coli MG1655. First, lacZ and wcaJ, two competitive pathway genes, were disrupted to block the invalid consumption of lactose and GDP-fucose, respectively. Next, the lacY gene was strengthened by switching its native promoter to PJ23119. To enhance the supply of endogenous GDP-fucose, the promoters of gene clusters manC-manB and gmd-fcl were strengthened individually or in combination. Subsequently, chromosomal integration of a constitutive PJ23119 promoter-based BKHT expression cassette (PJ23119-BKHT) was performed in the arsB and recA loci. The most productive plasmid-based and plasmid-free strains produced 76.9 and 50.1 g/L 2'-FL by fed-batch cultivation, respectively. Neither of them generated difucosyl lactose nor 3-fucosyllactose as byproducts.

At-Home, Cell-Free Synthetic Biology Education Modules for Transcriptional Regulation and Environmental Water Quality Monitoring.

As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by simply adding water and DNA to freeze-dried crude extracts of non-pathogenic Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a "build-your-own" activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high-school students in their classrooms─and at home─without professional laboratory equipment. This work promises to catalyze access to interactive synthetic biology education opportunities.

La(Ca)CrO3-Filled SiCN Precursor Thin Film Temperature Sensor Capable to Measure up to 1100 °C High Temperature.

Thin-film sensors are regarded as advanced technologies for in situ condition monitoring of components operating in harsh environments, such as aerospace engines. Nevertheless, these sensors encounter challenges due to the high-temperature oxidation of materials and intricate manufacturing processes. This paper presents a simple method to fabricate high temperature-resistant oxidized SiCN precursor and La(Ca)CrO3 composite thin film temperature sensors by screen printing and air annealing. The developed sensor demonstrates a broad temperature response ranging from 200 °C to 1100 °C with negative temperature coefficients (NTC). It exhibits exceptional resistance to high-temperature oxidation and maintains performance stability. Notably, the sensor's resistance changes by 3% after exposure to an 1100 °C air environment for 1 h. This oxidation resistance improvement surpasses the currently reported SiCN precursor thin-film sensors. Additionally, the sensor's temperature coefficient of resistance (TCR) can reach up to -7900 ppm/°C at 200 °C. This strategy is expected to be used for other high-temperature thin-film sensors such as strain gauges, heat flux sensors, and thermocouples. There is great potential for applications in high-temperature field monitoring.

Exsolution of Iron Nanoparticles from LCTFeO3 for Oxygen Evolution Reaction

Perovskite oxides (ABO3) with stable structures and flexible compositions are considered to be alternative catalysts for the oxygen evolution reaction (OER). With A-site deficiency in a perovskite, the doping metals in the B-site may be exsolved by calcination in a reduction atmosphere, which is a promising approach to increasing the activity of the catalyst. Here, an iron doped La(0.2+x)Ca(0.7-x)Ti(1-x)FexO3 (x = 0.05, 0.3, LCTFex) perovskite is successfully synthesized by a mild sol-gel method, and the iron nanoparticles anchored perovskite (R-LCTFex) can be fabricated after reduction under 5% H2/N2 atmosphere in a tubular furnace, which can be seen clearly in SEM and STEM images. Notably, the onset overpotential of R-LCTFe0.3 is reduced by 130 mV compared with LCTFe0.3 and the current density of R-LCTFe0.3 is 12-fold higher than LCTFe0.3 at the overpotential of 530 mV. The enhanced OER performance is triggered by the exsolution of Fe nanoparticles. This study establishes that exsolution of metal nanoparticles from a perovskite is a useful tactic for enhancing OER performance.

Preferential sugar utilization by bifidobacterial species.

Aim: Bifidobacteria benefit host health and homeostasis by breaking down diet- and host-derived carbohydrates to produce organic acids in the intestine. However, the sugar utilization preference of bifidobacterial species is poorly understood. Thus, this study aimed to investigate the sugar utilization preference (i.e., glucose or lactose) of various bifidobacterial species. Methods: Strains belonging to 40 bifidobacterial species/subspecies were cultured on a modified MRS medium supplemented with glucose and/or lactose, and their preferential sugar utilization was assessed using high-performance thin-layer chromatography. Comparative genomic analysis was conducted with a focus on genes involved in lactose and glucose uptake and genes encoding for carbohydrate-active enzymes. Results: Strains that preferentially utilized glucose or lactose were identified. Almost all the lactose-preferring strains harbored the lactose symporter lacS gene. However, the comparative genomic analysis could not explain all their differences in sugar utilization preference. Analysis based on isolate source revealed that all 10 strains isolated from humans preferentially utilized lactose, whereas all four strains isolated from insects preferentially utilized glucose. In addition, bifidobacterial species isolated from hosts whose milk contained higher lactose amounts preferentially utilized lactose. Lactose was also detected in the feces of human infants, suggesting that lactose serves as a carbon source not only for infants but also for gut microbes in vivo. Conclusion: The different sugar preference phenotypes of Bifidobacterium species may be ascribed to the residential environment affected by the dietary habits of their host. This study is the first to systematically evaluate the sugar uptake preference of various bifidobacterial species.

Expression of His-tagged NADPH-dependent acetoacetyl-CoA reductase in recombinant Escherichia coli BL-21(DE3)

Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E.coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4h, induction time of 12h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 Umg-1 protein and total protein concentration of 0.518mgmg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 Umg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.

Comparative genomics of Cronobacter sakazakii strains from a powdered infant formula plant reveals evolving populations

Cronobacter sakazakii is an opportunistic pathogen causing life-threatening infections in newborns. C. sakazakii strains (N = 26) isolated from a powdered infant formula (PIF) plant over a 15-years period were sequenced. Seventeen strains belonged to sequence type ST83 and nine to ST4. Core-genome MLST revealed clonality of most ST83 and some ST4 strains, showing that these stains derive from persistent populations. A genome comparison between the ST83 and ST4 strains identified population-specific genes, of which some are potentially involved in persistence, such as aggregation and stress-resistance genes in ST83 and fimbrial genes in ST4 strains.A single nucleotide polymorphisms comparison revealed 20 SNPs in a lactose operon. This lactose operon was shown to be transposon associated and was present in all PIF isolates, yet only in 171 of 449 public available C. sakazakii genomes, suggesting a role for lactose as evolutionary force in a PIF environment.Eighteen and 20 antimicrobial resistance genes were found in the ST83 and ST4 strain set, respectively. Four ST4 strains possessed a mcr6.1 gene acquired via HGT. encoding colistin resistance.We show that evolving C. sakazakii populations persist concomitantly in an industrial environment, which has a potential impact on public health.

Designing glucose utilization "highway" for recombinant biosynthesis

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae.

Sacchromycescerevisiae Peptidyl-prolylcis/trans-isomerase Rrd1 has been linked to DNA repair, bud morphogenesis, advancement of the G1 phase, DNA replication stress, microtubule dynamics and is also necessary for the quick decrease in Sgs1p levels in response to rapamycin. In present study, Rrd1 gene was amplified by standard PCR and subsequently cloned downstream to bacteriophage T7 inducible promoter and lac operator of expression vector pET21d(+). Additionally, immobilized metal affinity chromatography (IMAC) was used to purify the protein upto its homogeneity, and its homogeneous purity was further confirmed through western blotting. Size exclusion chromatography implies that Rrd1 is existing as monomer in its natural state. Foldwise Rrd1 protein belongs to PTPA-like protein superfamily. Rrd1 showed characteristic negative minima at 222 and 208 nm represent protein typically acquired α helix in the far-UV CD spectra. Fluorescence spectra showed properly folded tertiary structures of Rrd1 at physiological conditions. Rrd1protein can be identified from different species using a fingerprint created by PIPSA analysis. The protein's abundance could aid in its crystallization, biophysical characterization and identification of other-interacting partners of Rrd1 protein.

The impact of metabolism on the adaptation of organisms to environmental change.

Since Jacob and Monod's discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as "metabolic reprogramming." The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of E. coli to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible.

A unified transform method for general robust property of probabilistic Boolean control networks

This article proposes a unified transform method for general robust property of probabilistic Boolean control networks (PBCNs). Here, the general robust property of PBCNs means any given robust property of any given PBCN with probability one. Specifically, the method is that the investigation on any given robust property of any given PBCN with probability one can be equivalently transformed into the investigation on the given robust property of the corresponding Boolean control network (BCN). Note that the PBCN has time-varying probability distribution and the property can be set according to the needs of specific problems. As an application of the method, we study the robust set stabilization with probability one of PBCNs with positive time-varying probability distribution. We derive the results through a discussion of the robust set stabilization of the corresponding BCNs. Also, we characterize all of the time-optimal robust set stabilizers of the PBCNs. A reduced disturbed PBCN model of the lac operon in the Escherichia coli is provided to show the effectiveness of the achieved results.

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system.

Current research focuses on the soluble and high-level expression of biologically active recombinant human IL-29 protein inEscherichia coli. The codon-optimized IL-29 gene was cloned into the Champion™ pET SUMO expression system downstream of the SUMO tag under the influence of the T7 lac promoter. The expression of SUMO-fused IL-29 protein was compared in E. coli Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami 2(DE3). The release of the SUMO fusion partner resulted in approximately 98mg of native rhIL-29 protein with a purity of 99% from 1l of fermentation culture. Purified rhIL-29 was found to be biologically active, as evaluated by its anti-proliferation assay. It was found that Champion™ pET SUMO expression system can be used to obtained high yield of biologically active soluble recombinant human protein compared to other expression vector.

Maximum-Likelihood State Estimators in Probabilistic Boolean Control Networks.

This study addresses state estimation problems for probabilistic Boolean control networks (PBCNs). Compared with deterministic Boolean networks, PBCNs have the stochastic switching in logical update functions in the state equation. Consequently, statistical analysis is required to estimate unavailable states, which induces an optimization problem called maximum-likelihood estimation. This article mainly focuses on two scenarios: 1) state estimation from partially measured state and 2) state estimation from output data, meaning observer design. The resulting optimization problems are solved using efficient algorithms based on dynamic programming. Concurrently, Dijkstra-type algorithms, which solve equivalent shortest path problems, are also proposed using best-first search. Furthermore, both the proposed algorithms derive novel observer design methods for PBCNs. The proposed algorithms are evaluated with practical estimation problems aiming to the sensor reduction and applied to gene regulatory networks of apoptosis and Lac operon.

Exsolution of Iron Nanoparticles from LCTFeO3 for Oxygen Evolution Reaction

Perovskite oxides (ABO3) with stable structures and flexible compositions are considered to be alternative catalysts for the oxygen evolution reaction (OER). With A-site deficiency in a perovskite, the doping metals in the B-site may be exsolved by calcination in a reduction atmosphere, which is a promising approach to increasing the activity of the catalyst. Here, an iron doped La(0.2+x)Ca(0.7-x)Ti(1-x)FexO3 (x = 0.05, 0.3, LCTFex) perovskite is successfully synthesized by a mild sol-gel method, and the iron nanoparticles anchored perovskite (R-LCTFex) can be fabricated after reduction under 5% H2/N2 atmosphere in a tubular furnace, which can be seen clearly in SEM and STEM images. Notably, the onset overpotential of R-LCTFe0.3 is reduced by 130 mV compared with LCTFe0.3 and the current density of R-LCTFe0.3 is 12-fold higher than LCTFe0.3 at the overpotential of 530 mV. The enhanced OER performance is triggered by the exsolution of Fe nanoparticles. This study establishes that exsolution of metal nanoparticles from a perovskite is a useful tactic for enhancing OER performance.

Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray.

Among the wheat biotic stresses, Sitobion avenae is one of the main factors devastating the wheat yield per hectare. The study's objective was to find out the laccase (lac) efficacy; as a potential RNAi target against grain aphids. The Sitobion avenae lac (Salac) was confirmed by Reverse Transcriptase-PCR. Gene was sequenced and accession number "ON703252" was allotted by GenBank. ERNAi tool was used to design 143 siRNA and one dsRNA target. 69% mortality and 61% reduction in lac expression were observed 8D-post lac DsRNA feeding. Phylogenetic analysis displayed the homology of grain aphid lac gene with peach potato, pea, and Russian wheat aphids. While Salac protein was found similar to the Russian grain, soybean, pea, and cedar bark aphid lac protein multi-copper oxidase. The dsRNAlac spray-induced silencing shows systematic translocation from leaf to root; with maximum lac expression found in the root, followed by stem and leaf 9-13D post-spray; comparison to control. RNAi-GG provides the Golden Gate cloning strategy with a single restriction ligation reaction used to achieve lac silencing. Agrobacterium tumefaciens mediated in planta and in-vitro transformation was used in the study. In vitro transformation, Galaxy 2012 yielded a maximum transformation efficiency (1.5%), followed by Anaj 2017 (0.8%), and Punjab (0.2%). In planta transformation provides better transformation efficiencies with a maximum in Galaxy 2012 (16%), and a minimum for Punjab (5%). Maximum transformation efficiency was achieved for all cultivars with 250 μM acetosyringone and 3h co-cultivation. Galaxy 2012 exhibited maximum transformation efficiency, and aphid mortality post-feeding transgenic wheat.

Bacterial cellulose: A highly versatile nanomaterial.

Bacterial cellulose: A highly versatile nanomaterial.

Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses

BackgroundLaccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide.ResultsIn total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron–exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants.ConclusionsThis study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Spatiotemporal Gene Expression by a Genetic Circuit for Chemical Production in Escherichia coli.

Gene expression in spatiotemporal distribution improves the ability of cells to respond to changing environments. For microbial cell factories in artificial environments, reconstruction of the target compound's biosynthetic pathway in a new spatiotemporal dimension/scale promotes the production of chemicals. Here, a genetic circuit based on the Esa quorum sensing and lac operon was designed to achieve the dynamic temporal gene expression. Meanwhile, the pathway was regulated by an l-cysteine-specific sensor and relocalized to the plasma membrane for further flux enhancement to l-cysteine and toxicity reduction on a spatial scale. Finally, the integrated spatiotemporal regulation circuit for l-cysteine biosynthesis enabled a 14.16 g/L l-cysteine yield in Escherichia coli. Furthermore, this spatiotemporal regulation circuit was also applied in our previously constructed engineered strain for pantothenic acid, methionine, homoserine, and 2-aminobutyric acid production, and the titer increased by 29, 33, 28, and 41%, respectively. These results highlighted the applicability of our spatiotemporal regulation circuit to enhance the performance of microbial cell factories.

Construction of the advanced flavin mononucleotide producers in the flavinogenic yeast Candida famata.

Flavin mononucleotide (FMN, riboflavin-5'-phosphate) is flavin coenzyme synthesized in all living organisms from riboflavin (vitamin B2 ) after phosphorylation in the reaction catalyzed by riboflavin kinase. FMN has several applications mostly as yellow colorant in food industry due to 200 times better water solubility as compared to riboflavin. Currently, FMN is produced by chemical phosphorylation of riboflavin, however, final product contains up to 25% of flavin impurities. Microbial overproducers of FMN are known, however, they accumulate this coenzyme in glucose medium. Current work shows that the recombinant strains of the flavinogenic yeast Candida famata with overexpressed FMN1 gene coding for riboflavin kinase in the recently isolated by us advanced riboflavin producers due to overexpression of the structural and regulatory genes of riboflavin synthesis and of the putative exporter of riboflavin from the cell, synthesized elevated amounts of FMN in the media not only with glucose but also in lactose and cheese whey. Activation of FMN accumulation on lactose and cheese whey was especially strong in the strains which expressed the gene of transcription activator SEF1 under control of the lactose-induced LAC4 promoter. The accumulation of this coenzyme by the washed cells of the best recombinant strain achieved 540 mg/L in the cheese whey supplemented only with ammonium sulfate during 48 h in shake flask experiments.

Unlocking the strength of inducible promoters in Gram-negative bacteria.

Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the -10 and -35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus -10 and -35 sequence boxes strongly targeted by σ70 , incorporation of a strong ribosome binding site recognized broadly by Gram-negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which significantly improves the signal-to-background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.