Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Metabolic engineering of genome-streamlined strain Pseudomonas putida KTU-U27 for medium-chain-length polyhydroxyalkanoate production from xylose and cellobiose.

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-Ptac-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.

Creating an efficient 1,2-dichloroethane-mineralizing bacterium by a combination of pathway engineering and promoter engineering.

Currently, 1,2-dichloroethane (DCA) is frequently detected in groundwater and has been listed as a potential human carcinogen by the U.S. EPA. Owing to its toxicity and recalcitrant nature, inefficient DCA mineralization has become a bottleneck of DCA bioremediation. In this study, the first engineered DCA-mineralizing strain KTU-P8DCA was constructed by functional assembly of DCA degradation pathway and enhancing pathway expression with a strong promoter P8 in the biosafety strain Pseudomonas putida KT2440. Strain KTU-P8DCA can metabolize DCA to produce CO2 and utilize DCA as the sole carbon source for cell growth by quantifying 13C stable isotope ratios in collected CO2 and in lyophilized cells. Strain KTU-P8DCA exhibited superior tolerance to high concentrations of DCA. Excellent genetic stability was also observed in continuous passage culture. Therefore, strain KTU-P8DCA has enormous potential for use in bioremediation of sites heavily contaminated with DCA. In the future, our strategy for pathway construction and optimization is expected to be developed as a standard pipeline for creating a wide variety of new contaminants-mineralizing microorganisms. The present study also highlights the power of synthetic biology in creating novel degraders for environmental remediation.

[Microwave sensor for recognition of abnormal nodule tissue on body surface].

For the detection and identification of abnormal nodular tissues on the body surface, a microwave sensor structure loaded with a spiral resonator is proposed in this paper, a sensor simulation model is established using HFSS software, the structural parameters are optimized, and the actual sensor is fabricated. The S21 parameters of the tissue were obtained when nodules appeared by simulation, and the characteristic relationship between the difference of S21 parameters with position was analyzed and tested experimentally. The results showed that when nodules were present in normal tissues, the curve of S21 parameter difference with position change had obvious inverted bimodal characteristics, and the extreme value of S21 parameter difference appeared when the sensor was directly above the nodules, which was easy to identify the position of nodules. It provides an objective detection tool for the identification of abnormal nodular tissues on the body surface.

Unsterile production of a polyhydroxyalkanoate copolymer by Halomonas cupida J9.

Microbial production of bioplastics polyhydroxyalkanoates (PHA) has opened new avenues to resolve "white pollution" caused by petroleum-based plastics. PHAs consisting of short- and medium-chain-length monomers, designated as SCL-co-MCL PHAs, exhibit much better thermal and mechanical properties than PHA homopolymers. In this study, a halophilic bacterium Halomonas cupida J9 was isolated from highly saline wastewater and proven to produce SCL-co-MCL PHA consisting of 3-hydroxybutyrate (3HB) and 3-hydroxydodecanoate (3HDD) from glucose and glycerol. Whole-genome sequencing and functional annotation suggest that H. cupida J9 may possess three putative PHA biosynthesis pathways and a class I PHA synthase (PhaCJ9). Interestingly, the purified His6-tagged PhaCJ9 from E. coli BL21 (DE3) showed polymerizing activity towards 3HDD-CoA and a phaCJ9-deficient mutant was unable to produce PHA, which indicated that a low-substrate-specificity PhaCJ9 was exclusively responsible for PHA polymerization in H. cupida J9. Docking simulation demonstrated higher binding affinity between 3HB-CoA and PhaCJ9 and identified the key residues involved in hydrogen bonds formation between 3-hydroxyacyl-CoA and PhaCJ9. Furthermore, His489 was identified by site-specific mutagenesis as the key residue for the interaction of 3HDD-CoA with PhaCJ9. Finally, PHA was produced by H. cupida J9 from glucose and glycerol in shake flasks and a 5-L fermentor under unsterile conditions. The open fermentation mode makes this strain a promising candidate for low-cost production of SCL-co-MCL PHAs. Especially, the low-specificity PhaCJ9 has great potential to be engineered for an enlarged substrate range to synthesize tailor-made novel SCL-co-MCL PHAs.

Enhanced production of polyhydroxyalkanoates in Pseudomonas putida KT2440 by a combination of genome streamlining and promoter engineering.

Polyhydroxyalkanoates (PHAs), a class of bioplastics produced by a variety of microorganisms, have become the ideal alternatives for oil-derived plastics due to their superior physicochemical and material characteristics. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from various substrates. In this study, a novel strategy of the large-scale deletion of genomic islands (GIs) coupling with promoter engineering was developed in P. putida KT2440 for constructing the minimal genome cell factories (MGF) capable of efficiently producing mcl-PHA. Firstly, P. putida KTU-U13, a 13 GIs- and upp-deleted mutant derived from the parental strain P. putida KT2440, was used as a starting strain for further deletion of GIs to generate a series of genome-reduced strains. Subsequently, the two minimal genome strains KTU-U24 and KTU-U27, which had a 7.19% and 8.35% reduction relative to the genome size of KT2440 and were advantageous over the strain KTU (KT2440∆upp) and KTU-U13 in several physiological traits such as the maximum specific growth rate, plasmid transformation efficiency, heterologous protein expression capacity and PHA production capacity, were selected as the chassis cells for PHA metabolic engineering. To prevent the formation of the by-product gluconic acid, the glucose dehydrogenase gene was deleted in KTU-U24 and KTU-U27, resulting in KTU-U24∆gcd and KTU-U27∆gcd. To enhance the transcriptional level of PHA synthase genes (phaC) and the supply of the precursor acetyl-CoA, a strong endogenous promoter P46 was inserted into upstream of the phaC operon and pyruvate dehydrogenase gene in the genome of KTU-U24∆gcd and KTU-U27∆gcd, to generate KTU-U24∆gcd-P46CA and KTU-U27∆gcd-P46CA, with the PHA yield of 50.5 wt% and 53.8 wt% (weight percent of PHA in cell dry weight). Finally, KTU-U27∆gcd-P46CA, the most minimal KT2440 chassis currently available, was able to accumulate the PHA to 55.82 wt% in a 5-l fermentor, which is the highest PHA yield obtained with P. putida KT2440 so far. This study suggests that genome streamlining in combination with promoter engineering may be a feasible strategy for the development of the MGF for the efficient production of high value products. Moreover, further streamlining of the P. putida KT2440 genome has great potential to create the optimal chassis for synthetic biology applications.

Flexible Microwave Biosensor for Skin Abnormality Detection Based on Spoof Surface Plasmon Polaritons.

Point-of-care testing plays an important role in the detection of skin abnormalities. The detection of skin abnormalities requires sufficient depth and no harm. A flexible microwave biosensor based on spoof surface plasmon polaritons was designed to meet the requirements of skin abnormalities. The designed biosensor, which works at 11.3 GHz, is small and can be flexibly attached to the skin surface of any part of the human body for measurement. The health status of the skin can be evaluated by the resonant frequency and the magnitude of the reflection coefficient of the sensor. The sensor was tested on pork skin. The experiment results showed that the sensor can detect skin abnormalities such as skin burn, skin tumor, and others. Compared with other sensors, the sensor has sufficient penetration depth because of the strong penetration of microwave electromagnetic waves. It is the first flexible microwave biosensor used for skin, which involves point-of-care testing, and continuous monitoring of skin.

High-resolution probe design for measuring the dielectric properties of human tissues.

BACKGROUND: In order to use the microwave to measure the dielectric constant of the human body and improve the measurement resolution, a small near-field probe working at 915 MHz is designed in this paper. METHOD: Based on the electric small loop antenna model loaded by the spiral resonator (SR), a small near-field probe was designed. The probe model is designed and optimized by the HFSS (high frequency structure simulator) software. The human tissues were tested by the manufactured probe and the relationship between the S11 parameters of the probe and the human tissues was analyzed. RESULTS AND CONCLUSIONS: A probe with small size was designed and fabricated, with the overall size of 10.0 mm × 12.0 mm × 0.8 mm. The probe has a good performance with a 30.7 dB return loss, a 20 MHz bandwidth at the resonance point, and a distance resolution of 10 mm. Due to the small size and good resolution of the probe, it can be used in the measurement of human tissues.

The Role of a New Compound Micronutrient Multifunctional Fertilizer against Verticillium dahliae on Cotton.

Verticillium dahliae Kleb., the causal pathogen of vascular wilt, can seriously reduce the yield and quality of many crops, including cotton (Gossypium hirsutum). To control the harm caused by V. dahliae, considering the environmental pollution of chemical fungicides and their residues, the strategy of plant nutrition regulation is becoming increasingly important as an eco-friendly method for disease control. A new compound micronutrient fertilizer (CMF) found in our previous study could reduce the damage of cotton Verticillium wilt and increase yield. However, there is little information about the mode of action of CMF to control this disease. In the present study, we evaluated the role of CMF against V. dahliae and its mechanism of action in vitro and in vivo. In the laboratory tests, we observed that CMF could inhibit hyphal growth, microsclerotia germination, and reduce sporulation of V. dahliae. Further studies revealed that the biomass of V. dahliae in the root and hypocotyl of cotton seedlings treated with CMF were significantly reduced compared with the control, and these results could explain the decline in the disease index of cotton Verticillium wilt. Furthermore, those key genes involved in the phenylpropanoid metabolism pathway, resistance-related genes defense, and nitric oxide signaling pathway were induced in cotton root and hypocotyl tissue when treated with CMF. These results suggest that CMF is a multifaceted micronutrient fertilizer with roles in inhibiting the growth, development, and pathogenicity of V. dahliae and promoting cotton growth.

Wide range temperature-dependent deformation and fracture mechanisms for 8701 under dynamic and static loading.

Although the RDX-based composite explosive 8701 explosive 8701 has been widely used to achieve military goals, its mechanical properties have not been carefully investigated. In the present study, we focused on the mechanical response of 8701 at a wide range temperature from -125 °C to 100 °C under both quasi-static (about 0.001 s-1) and high-rate compression loading (about 600 s-1). The stress-strain curves exhibit different tendencies at different temperatures for both quasi-static and high strain-rate loading. The failure stress and elastic/storage modulus present important temperature-dependence. Differential scanning calorimetry (DSC) tests showed that the glass transition temperature and softening temperature of 8701 are 11.61 °C and 15.14 °C respectively, which is lower than that of the binder (with glass transition temperature of 25 °C and softening temperature 38 °C). For the quasi-static loading, scanning electron microscopy (SEM) observations revealed that 8701 shows an interface debonding failure mode along the binder phase below 15 °C, while the mechanical behavior of 8701 is dominated by softening behavior of the binder above 38 °C. For high-rate loading, 8701 shows a mixture of interface debonding and trans-granular cleavage when below 15.14 °C.