Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.

Study on the detection of anthrax by ICP-MS based on gold nanoparticle labeling.

BACKGROUND: In recent years, inductively coupled plasma mass spectrometry (ICP-MS) has been widely used in the field of molecular biology because of its unique advantages. Anthrax is a widespread and long-standing infectious disease, which affects and restricts people's work and life seriously. OBJECTIVE: The study goal is to develop a new method for the detection of anthrax. METHODS: A rapid, sensitive and accurate method for the detection of anthrax characteristic DNA was proposed by combing gold nanoparticles (AuNPs) and inductively coupled plasma mass spectrometry. RESULTS: The linear range of this method is 100-2500 pmol/L and the limit of detection of 16.61 pmol/L. CONCLUSION: The proposed method has numerous advantages, including simplicity of operation, high sensitivity, and specificity, which provides a new idea for the detection of anthrax. Importantly, this methodology has good potential for the detection of other biological substances such as bacteria and viruses by changing the modification sequence on the nanoparticle probe.

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris.

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6 U ml(-1) (batch cultivation with flask cultivation) or 14.4 U ml(-1) (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.

Significant Reduction of Interfacial Thermal Resistance and Phonon Scattering in Graphene/Polyimide Thermally Conductive Composite Films for Thermal Management.

The developing flexible electronic equipment are greatly affected by the rapid accumulation of heat, which is urgent to be solved by thermally conductive polymer composite films. However, the interfacial thermal resistance (ITR) and the phonon scattering at the interfaces are the main bottlenecks limiting the rapid and efficient improvement of thermal conductivity coefficients (λ) of the polymer composite films. Moreover, few researches were focused on characterizing ITR and phonon scattering in thermally conductive polymer composite films. In this paper, graphene oxide (GO) was aminated (NH2-GO) and reduced (NH2-rGO), then NH2-rGO/polyimide (NH2-rGO/PI) thermally conductive composite films were fabricated. Raman spectroscopy was utilized to innovatively characterize phonon scattering and ITR at the interfaces in NH2-rGO/PI thermally conductive composite films, revealing the interfacial thermal conduction mechanism, proving that the amination optimized the interfaces between NH2-rGO and PI, reduced phonon scattering and ITR, and ultimately improved the interfacial thermal conduction. The in-plane λ (λ ||) and through-plane λ (λ ⊥) of 15 wt% NH2-rGO/PI thermally conductive composite films at room temperature were, respectively, 7.13 W/mK and 0.74 W/mK, 8.2 times λ || (0.87 W/mK) and 3.5 times λ ⊥ (0.21 W/mK) of pure PI film, also significantly higher than λ || (5.50 W/mK) and λ ⊥ (0.62 W/mK) of 15 wt% rGO/PI thermally conductive composite films. Calculation based on the effective medium theory model proved that ITR was reduced via the amination of rGO. Infrared thermal imaging and finite element simulation showed that NH2-rGO/PI thermally conductive composite films obtained excellent heat dissipation and efficient thermal management capabilities on the light-emitting diodes bulbs, 5G high-power chips, and other electronic equipment, which are easy to generate heat severely.

[Effect of transglutaminase-gene disruption in Streptomyces hygroscopicus on cell differentiation].

OBJECTIVE: To provide theoretical basis for understanding the physiological function of transglutaminase in Streptomyces, which would benefit for improving the production of this enzyme, transglutaminase gene was disrupted in Streptomyces hygroscopicus. METHODS: First, the interruption plasmid pKC1139-TG1 was constructed by inserting a segment of transglutaminase gene in the temperature-sensitive vector pKC1139. Second, pKC1139-TG1 was transformed into S. hygroscopicus protoplast and the interruption plasmid was inserted into the chromosome by homogenous recombination. Third, the strain with disrupted transglutaminase gene was screened by apramycin resistance and named as S. h-delta TG. RESULTS: Compared with the parent strain, S. h-delta TG was able to grow as substrate mycelium, but not able to form aerial hyphae. CONCLUSION: Transglutaminase may be involved in the aerial hyphae formation of S. hygroscopicus.

Highly Thermal Conductivities, Excellent Mechanical Robustness and Flexibility, and Outstanding Thermal Stabilities of Aramid Nanofiber Composite Papers with Nacre-Mimetic Layered Structures.

Aramid nanofiber (ANF) paper has shown potential applications in flexible electronics. However, its inherent low thermal conductivity coefficient (λ) values might threaten the safety of devices under a high-power working condition. In this work, polydopamine-functionalized boron nitride nanosheet (BNNS@PDA)/ANF thermally conductive composite papers with nacre-mimetic layered structures were prepared via highly efficient vacuum-assisted filtration followed by hot pressing. For a given BNNS loading, the surface functionalization of BNNS could further enhance the thermal conductivities and mechanical properties of BNNS@PDA/ANF composite papers. BNNS@PDA/ANF composite papers presented anisotropic thermal conductivities, and the through-plane (λ⊥) and in-plane (λ∥) values of the 50 wt % BNNS@PDA/ANF composite papers reached 0.62 and 3.94 W/mK, 181.8 and 196.2% higher than those of original ANF paper, respectively, which were also higher than those of 50 wt % BNNS/ANF composite papers (λ⊥ = 0.52 W/mK and λ∥ = 3.33 W/mK). The tensile strength of the 50 wt % BNNS@PDA/ANF composite papers reached 36.8 MPa, 30.5% higher than that of 50 wt % BNNS/ANF composite papers (28.2 MPa). In addition, the heat resistance index (THRI) of the 50 wt % BNNS@PDA/ANF composite papers was further increased to 223.1 °C. Overall, our fabricated BNNS@PDA/ANF composite papers possess highly thermal conductivities, excellent mechanical robustness and flexibility, and outstanding thermal stabilities, showing great potential applications in the fields of intelligent wearable equipment, flexible supercapacitors, and flexible electronics.

[Heterologous expression and enzymatic analysis of Streptomyces griseus trypsin in Streptomyces lividans].

Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.