Multiscale engineering of microbial cell factories: A step forward towards sustainable natural products industry.

Microbial cell factories (bacteria and fungi) are the leading producers of beneficial natural products such as lycopene, carotene, herbal medicine, and biodiesel etc. These microorganisms are considered efficient due to their effective bioprocessing strategy (monoculture- and consortial-based approach) under distinct processing conditions. Meanwhile, the advancement in genetic and process optimization techniques leads to enhanced biosynthesis of natural products that are known functional ingredients with numerous applications in the food, cosmetic and medical industries. Natural consortia and monoculture thrive in nature in a small proportion, such as wastewater, food products, and soils. In similitude to natural consortia, it is possible to engineer artificial microbial consortia and program their behaviours via synthetic biology tools. Therefore, this review summarizes the optimization of genetic and physicochemical parameters of the microbial system for improved production of natural products. Also, this review presents a brief history of natural consortium and describes the functional properties of monocultures. This review focuses on synthetic biology tools that enable new approaches to design synthetic consortia; and highlights the syntropic interactions that determine the performance and stability of synthetic consortia. In particular, the effect of processing conditions and advanced genetic techniques to improve the productibility of both monoculture and consortial based systems have been greatly emphasized. In this context, possible strategies are also discussed to give an insight into microbial engineering for improved production of natural products in the future. In summary, it is concluded that the coupling of genomic modifications with optimum physicochemical factors would be promising for producing a robust microbial cell factory that shall contribute to the increased production of natural products.

Design and Fabrication of a Microfluidic Chip for Particle Size-Exclusion and Enrichment.

Based on the size of particles, a microfluidic chip integrating micro particles capture, controlled release and counting analysis was designed and fabricated in this paper. The chip is composed of a polydimethylsiloxane (PDMS) cover sheet and a PDMS substrate. The PDMS substrate is made of a sample inlet, microfluidic channels, a micropillar array, a three-dimensional (3D) focusing channel, and a sample outlet. The chip was fabricated by the multistep SU-8 lithography and PDMS molding method in this study. The micropillar array and channels in the chip can be molded in one step and can be replicated multiple times, which reduces the production cost and increases the practicability of the chip. Using a homemade electromagnetic drive device, the detection function of the chip was tested using a deionized water solution containing 22 µm polyethylene particles. The results showed that under the action of electromagnetic force, the chip enriched polyethylene particles; when the electromagnetic force disappeared, the enriched polyethylene particles were released by injecting buffer solution, and it was looked at as new sample solution. The flow rate of the sample solution and the sheath flow solution (deionized water) was injected into the three-dimensional focusing channel at a flow rate ratio of 1:4, and the polyethylene particles sample solution was focused, which could be used for the counting and analysis of polyethylene particles. The work of this paper can provide a reference for the subsequent detection of circulating tumor cells (CTCs).

Rationally optimized generation of integrated Escherichia coli with stable and high yield lycopene biosynthesis from heterologous mevalonate (MVA) and lycopene expression pathways.

The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization. In the current study on taking lycopene biosynthesis as an example, an integrated Escherichia coli system has been generated successfully, which resulted into stable and high lycopene production. In this process, two modules of mevalonate (MVA) pathway and one module of lycopene expression pathway were completely integrated in the chromosome. Firstly, the copy number and integrated position of three modules of heterologous pathways were rationally optimized. Later, a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411. The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L (49.9 mg/g DCW) in a 5 L fermenter with mean productivity of 61.0 mg/L/h. Additionally, the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.

Ultrasonic thermometric measurement system for solid rocket combustion chambers.

Solid rocket motor (SRM) temperature is an important physical parameter for which there is no reliable in situ measurement device, apart from a thermocouple, for such a high temperature environment apart. In this study, an ultrasonic temperature measurement system was designed with an iridium-rhodium-40% alloy waveguide. Laboratory experiments showed that the device obtained ultrasonic signals up to 1800 °C with a temperature fitting curve from room temperature to 1800 °C. The thermometer also operated stably under high temperature and produced a repeatable calibration curve, at 97% repeatability. An error band was obtained with 95% confidence. At temperatures above 1000 °C, sensitivity gradually increased to a maximum of 0.0035 µs/°C. A corresponding application structure was established for an SRM before subjecting the sensor to a temperature test experiment. The temperature-time curve obtained detected a peak temperature at 1744 °C.

A novel plasmid-Escherichia coli system produces large batch dsRNAs for insect gene silencing.

BACKGROUND: RNA interference (RNAi)-based pest management requires efficient delivery and large-batch production of double-stranded (ds)RNA. We previously developed a nanocarrier-mediated dsRNA delivery system that could penetrate an insect's body and efficiently silence gene expression. However, there is a great need to improve the plasmid-Escherichia coli system for the mass production of dsRNA. Here, for efficient dsRNA production, we removed the rnc gene encoding endoribonuclease RNase III in E. coli BL21(DE3) and matched with the RNAi expression vector containing a single T7 promoter. RESULTS: The novel pET28-BL21(DE3) RNase III-system was successfully constructed to express vestigial (vg)-dsRNA against Harmonia axyridis. dsRNA was extracted and purified from cell cultures in four E. coil systems, and the yields of dsRNA in pET28-BL21(DE3) RNase III-, pET28-HT115(DE3), L4440-BL21(DE3) RNase III- and L4440-HT115(DE3) were 4.23, 2.75, 0.88 and 1.30 µg mL-1 respectively. The dsRNA expression efficiency of our novel E. coil system was three times that of L4440-HT115(DE3), a widely used dsRNA production system. The RNAi efficiency of dsRNA produced by our system and by biochemical synthesis was comparable when injected into Harmonia axyridis. CONCLUSION: Our system expressed dsRNA more efficiently than the widely used L4440-HT115(DE3) system, and the produced dsRNA showed a high gene-silencing effect. Notably, our pET28-BL21(DE3) RNase III-system provides a novel method for the mass production of dsRNA at low cost and high efficiency, which may promote gene function analysis and RNAi-based pest management. © 2020 Society of Chemical Industry.

Correction: An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome.

[This corrects the article DOI: 10.1371/journal.pone.0186891.].

A measurement system of high-temperature oxidation environment with ultrasonic Ir0.6Rth0.4 alloy thermometry.

Iridium-rhodium is generally applied as a thermocouple material, with max operating temperature about 2150 °C. In this study, a ultrasonic temperature measurement system was designed by using Iridium-rhodium (60%Ir-40%Rh) alloy as an acoustic waveguide sensor material, and the system was preliminarily tested in a high-temperature oxidation environment. The result of ultrasonic temperature measurement shows that this system can indeed work stably in high-temperature oxidation environments. The relationship between temperature and delay time of ultrasonic thermometry up to 2200 °C was illustrated. Iridium-rhodium materials were also investigated in order to fully elucidate the proposed waveguide sensor's performance in a high-temperature oxidation environment. This system lays a foundation for further application of high-temperature measurement.

Rapid and Efficient Collection of Platinum from Karstedt's Catalyst Solution via Ligands-Exchange-Induced Assembly.

Reported herein is a novel strategy for the rapid and efficient collection of platinum from Karstedt's catalyst solution. By taking advantage of a ligand-exchange reaction between alkynols and the 1,3-divinyltetramethyldisiloxane ligand (MViMVi) that coordinated with platinum (Pt(0)), the Karstedt's catalyst particles with a size of approximately 2.5 ± 0.7 nm could be reconstructed and assembled into larger particles with a size of 150 ± 35 nm due to the hydrogen bonding between the hydroxyl groups of the alkynol. In addition, because the silicone-soluble MViMVi ligand of the Karstedt's catalyst was replaced by water-soluble alkynol ligands, the resultant large particles were readily dispersed in water, resulting in rapid, efficient, and complete collection of platinum from the Karstedt's catalyst solutions with platinum concentrations in the range from ∼20 000 to 0.05 ppm. Our current strategy not only was used for the rapid and efficient collection of platinum from the Karstedt's catalyst solutions, but it also enabled the precise evaluation of the platinum content in the Karstedt's catalysts, even if this platinum content was extremely low (i.e., 0.05 ppm). Moreover, these platinum specimens that were efficiently collected from the Karstedt's catalyst solutions could be directly used for the evaluation of platinum without the need for pretreatment processes, such as calcination and digestion with hydrofluoric acid, that were traditionally used prior to testing via inductively coupled plasma mass spectrometry in conventional methods.

Enhanced production of biosynthesized lycopene via heterogenous MVA pathway based on chromosomal multiple position integration strategy plus plasmid systems in Escherichia coli.

The multiple plasmid system, mostly relied, for heterogeneous gene expression, results in genetic instability and low mean productivity. To address this, an integration method was employed for investigating expression of heterogenous pathway in E. coli cells; where mevalonate upper pathway was found efficiently expressed. Subsequently, to improve lycopene production, chromosomal multiple position integration strategy was used to strengthen mevalonate upper pathway. Meanwhile, the plasmid system was employed for mevalonate lower pathway and lycopene pathway expression to finally generate the mutant D711 strain. Comparatively, highest level of 68.5 mg/L lycopene was produced by D711 outperforming its maximum average productivity of 2.85 mg/L/h with over 2-folds enhancement. In addition, lycopene level was almost 224 mg/L after optimization of induction time, which was 3.3-fold higher than standard control condition. Finally, expression Performance Parameter was developed for scoring mutants and evaluating these two strategies, indicating chromosomal multiple position integration strategy as more efficient approach.

pH-Responsive Porous Nanocapsules for Controlled Release.

In this work pH-responsive porous nanocapsules have been successfully prepared from a ternary graft copolymer, poly(glycidyl methacrylate)-g-[poly(2-cinnamoyloxyethyl methacrylate)-r-poly(ethylene glycol) methyl ether-r-poly(2-diethylaminoethyl methacrylate)] or PGMA-g-(PCEMA-r-MPEG-r-PDEAEMA). The graft copolymers were fabricated by grafting three types of polymer chains onto the backbone polymer by using click chemistry. These ternary copolymers underwent self-assembly to form vesicles in a DMF/water solvent mixture. While the MPEG chains served as the corona and stabilized the vesicles, the vesicle wall was composed of a dominant PCEMA continuous phase that was interspersed by PDEAEMA domains. After photo-cross-linking, the PDEAEMA domains were embedded in the structurally locked PCEMA wall. By decreasing the pH of the external solution, we were able to trigger the release of encapsulated pyrene due to the capsule wall becoming porous as a result of the PDEAEMA chains bearing positively charged amine groups stretching into the water. While these pH-responsive porous nanocapsules exhibited potential applications in drug delivery, detection and catalysis, the strategy reported in this contribution also represented a new paradigm for the design and preparation of other novel stimuli-responsive porous nanocapsules.

An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome.

The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4-104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3-100%. Additionally, the recombination efficiencies were 2.5-45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.

Ultrasonic Al2O3 Ceramic Thermometry in High-Temperature Oxidation Environment.

In this study, an ultrasonic temperature measurement system was designed with Al2O3 high-temperature ceramic as an acoustic waveguide sensor and preliminarily tested in a high-temperature oxidation environment. The test results indicated that the system can indeed work stably in high-temperature environments. The relationship between the temperature and delay time of 26 °C-1600 °C ceramic materials was also determined in order to fully elucidate the high-temperature oxidation of the proposed waveguide sensor and to lay a foundation for the further application of this system in temperatures as high as 2000 °C.