Selenium (Selenium,Se), as one of the essential trace elements, is closely related to human health.The content of selenium in 51% farmland is low, leading to the shortage of selenium in the food chain, resulting to the insufficient intake of selenium by local residents.The health risk caused by selenium deficiency has also become one of the micronutritional imbalance in China. The commonly used selenium supplements include food (meat, eggs, milk), selenium tablets and selenium-rich yeast.
In 1989, areas with low selenium content or selenium deficiency accounted for 72% of the total region, and now declined to 51% after years of governance, with too low selenium content accounted for about 26%.Northeast China (including the Taihang Mountains,
Qinling Mountains and loess) contains less than 0.200 mg/kg. National nutrition survey in China showed that 105 million people in 366 counties face adverse health effects on for selenium deficiency.It was also shown that the daily
selenium intake was lower than in 39 – 61% of Chinese residents, according to the WHO/FAO recommended values (26 – 34 μg/d).
According to statistics, the selenium content varies in the human diet while meat and seafood are considered the advantageous selenium supplement material because of their relatively high selenium content. The selenium intake of selenium in animals is
mainly based on plants that absorb nutrients from the soil and allocated feed feeding, while the content of selenium converted in the body is determined by the morphology of selenium. Due to the imbalance of selenium content in grain
crops in China, it also leads to the unbalanced distribution of selenium content in feed raw materials in China, that is, there are obvious regional differences and correlation. Therefore, soil selenium supplementation and animal feed
selenium supplement is very important, which is the way to further high selenium content in human body.
Moreover, the effects of different selenium supplements differ in reality, because the bioavailability of different forms of selenium is also different, and the effects of different selenium supplements show certain differences. At the same time, because selenium shows a beneficial to toxic property in a narrow concentration range, insufficient or excessive intake can lead to adverse reactions, which to solve and control is a tough challenge. At the same dose, monolithic nanoselenium (SeNPs) has the advantage of low toxicity and high bioavailability compared to highly toxic selenite (Se2O32-), which also makes it a hot spot on how nanoselenium is absorbed into meat, eggs and milk to further replace inorganic selenium as a human selenium filling agent.
Problems still exist. In nature, the synthesis of nanoparticles often requires physical and chemical approaches to solve due to low chemical yields. However, the use of high temperature and high pressure, acidic media, chemical raw materials during the preparation process, and the more toxic by-product production will pose a threat to the environment. Therefore, new approaches to explore clean, low-cost and environment-friendly synthetic nanoparticles are receiving increasing attention.
Nanoselenium, SeNPs, a kind of nano particle, hence appeared to be the inspiration of our team. It generally refers to the zero-valent plasic selenium of nanoscale (30-200 nm), with functional properties common to inorganic and organic selenium, and with
low toxicity, high safety, large than surface area and high surface activity. Therefore, nano-selenium is not only more easily absorbed and utilized by the human body, but also better exerts the functions such as antioxidant and immune
regulation.
When SeNPs exhibited stronger antioxidant free radical clearance (DPPH, ·OH, ROS) than Na2SeO3 at concentrations less than 0.5 mmol/L.And when the particle size is less than 150 nm, it has a better free radical clearance ability. Most importantly, SeNPs
can be directly transmitted to cells, without involving complex uptake pathway and energy loss to the host, thus more conducive to intestinal absorption, can be absorbed through two ways of cell gap and endocytosis two ways.
Therefore, application of nanoselenium to complementary animal nutrition to the bioavailability of trace elements is a reasonable choice for selenium supplementation, which can be applied to a better food-nutrition production system in market.
Expanded from our inspiration and expectation, the focus of our project is on the inclusion of the application of nanoselenium and the development of innovative nanoselenium mono-supplementation systems, particularly the possibility to explore a more efficient way of supplementation, which gives high bioavailability of this element and expects its controlled implementation in organisms if can be widely accepted and formed into a safe and stable system in the future.
We decided to create a new cell biocatalyst, constructed to display selenite reductase on the surface of probiotic EcN cells. Then to construct the new engineered bacteria that has the potential to reduce selenite to nano-selenium in the intestinal tract
to enhance the Selenium content in oragnisms by applying the Microbial cell surface display system that functions by combining specific exogenous functional proteins with anchor proteins and acting on the cell membrane or cell wall
surface of receptor microbes. In our project, using surface display techniques in synthetic organisms, protein SerV01 identified in S. aureus LZ-01 can effectively reduce selenite to SeNPs on the cell surface of E. coli 1917 cells.
SeNPs can be directly transmitted to cells, without involving complex uptake pathway and energy loss to the host, thus more conducive to intestinal absorption, can be absorbed through two ways of cell gap and endocytosis two ways.
We conducted microbial screening of the local selenium-metal mining area in Lanzhou and selected several six potential reducing competent strains
with high efficiency. We simultaneously tested its reduction ability to prove tetravalent selenium and explored the possible proteins acting in it, setting three parallel actions as controls. We then used bioinformatics to find the
functional reductase SerV01, which can efficiently reduce selenite in the most reducing bacteria LZ-01(S. aureus), exploring its homology, enzymatic activity mechanics and ligands. The N-terminal domain sequence of ice cryonuclease,
the carrier protein we chose, was fused with the INP-N-SerV01 peptide of the bioase reductant SerV01.
The stability of the passenger protein derived from microorganisms is affected by operating conditions during direct extraction and synthesis, and also causes huge economic costs. Therefore, for the purpose of the experiment, we modified the passenger protein to achieve the overexpression of homologous or heterologous proteins through the transformation of high-copy plasmids. This method not only reduces the cost, but also increases the utilization rate of passenger protein. The cell vector contains multiple cryptic plasmids that may have effects on subsequent live expression of enzymes. We tested that the plasmids pSB1A3 from iGEM data resource could be successfully converted into EcN, and we insert strong promoter and long gene sequence as intergenic region to ensure subsequent high quality overexpression of protein (2018IGEM Distribution Kit, containing the constitutive promoter Plac). In our project, ice riboprotein(INP-N) was specifically used to anchor the target genetoto form the fusion polypeptide INP-SerV01 that can effectively anchor on the cell outer membrane.
Because intestinal microorganisms usually play an important role in the REDOX of substrates and can lead to changes in metal valence states during the adsorption and detoxification of heavy metals, while among them, EcN, a type of non-pathogenic symbiotic gram-negative probiotics, has a good probiotic effect and is used for the treatment of various intestinal diseases because it does not secrete exotoxin. Moreover, it is the host strain with the dominant expression of foreign protein. Analysis of EcN’s genomic structure suggests that it lacks virulence factors such as α-hemolystin, P fiber adhesins and semi-crude lipopolysaccharide phenotypes. Its expression systems containing suitable factors (e. g., microtin, adhesin and iron absorption) contributes to the properties of its probiotical traits.
Thus we chose this strain as our cell vector. We then anchored to the extracellular membrane of probiotics EcN to construct whole cell biocatalyst EcN-IS, which finally reduced selenite on the surface of the cell membrane in the pariplasmic space by applying the Microbial Cell Surface Display System, a system that acts on the surface of the cell membrane or cell wall of the receptor microorganism by combining specific foreign functional proteins with anchorin. Fusion proteins can be directly expressed on the surface of host cells for practical application purposes and are often used to demonstrate the adsorption and removal of excessive and excessive heavy metals by metal binding proteins. Cell-surface display technology can also be used to enhance the stability of catalytic hydrolases to resist temperature changes, and the fusion proteins of surface display as catalysts can be recycled, which is cost-effective compared to the high recovery cost of immobilized enzymes.
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