With the rapid development of biological technology ，more and more engineering bacteria with multiple functions have been created, which can be applied to various fields such as industry production, agriculture, environment, diagnosis and therapy.

  Industrial biotechnology, represented by the successive listing of polylactide, biosteel, and polylactic acid, has set off the third wave of biotechnology industry at the turn of the century, and has attracted more and more attention from the government and the public.

  In January 2017, the National Development and Reform Commission issued the “The 13th five-year plan for biological industry development”, the planning is put forward to the biological manufacturing industry output value over one trillion yuan in 2020, bio-based products was 25%, the proportion in all chemical production and biological energy to replace fossil energy quantity more than 56 million tons of standard coal, in areas such as electricity, gas supply, heating, fuel to achieve the goal of full scale application.

  However, in spite of the high attention, the commercialization of industrial biotechnology is not as fast as we expected，one of the important limiting factors is the low bioprocessing efficiency during microbial fermentation. At present, one way to solve this problem is to achieve the technology of high-cell-density growth.At present, one way to solve this problem is to develop that can be achieved. Among them, the key factor limiting cell density in bioreactors is the availability of oxygen during the late phases of fermentation.^[1]

  The growth and metabolism of aerobic microorganisms both require oxygen. When the oxygen demand of bacteria is not met, the respiration of the bacteria is inhibited, thereby inhibiting growth, and even causing the accumulation of by-products. The cell metabolic rate will slow down, and the formation of metabolites will also be hindered.

    For aerobic fermentation, dissolved oxygen is usually both a nutrient and an environmental factor. The activity of aerobic microorganism enzymes is highly dependent on oxygen, and the production of some products requires the participation of oxygen.

  Compared with oxygen dissolved in pure water, the solubility of oxygen is significantly reduced in fermentation broth due to the presence of various dissolved nutrients, inorganic salts and metabolites of microorganisms. In addition, the concentration of dissolved oxygen varies with temperature, pressure and salt. In general, the higher the temperature, the more salt dissolved, the lower the dissolved oxygen in the water; The higher the pressure, the higher the dissolved oxygen in the water.

  At present, the lack of oxygen in the fermentation process is mainly caused by the following two factors.

  One is insufficient dissolved oxygen in the medium. Dissolved oxygen refers to the dissolved oxygen in the molecular state of water. Since oxygen is undissolved gas, under the condition of normal pressure, 25 ° C, oxygen solubility in the water in the air is only about 0.25 tendency for L. Therefore, if the outside world cannot supply oxygen in time during the fermentation process, the amount of dissolved oxygen in the water can only maintain a very short period of normal respiration of the bacteria, it will be exhausted. Therefore, it is necessary to constantly introduce sterile air into the fermentation system and further disperse it by stirring in the fermentation tank, so as to maintain a moderate concentration of dissolved oxygen in the fermentation liquid, but sometimes it is still unable to meet the oxygen demand of fermentation production. With the increase of fermentation tank volume, the raw material and power consumption of the fermentation process have increased to a large extent, and the demand for oxygen is also increasing.^[3]

  Now, most of the solutions to the problem of insufficient dissolved oxygen content are to reduce the temperature of the medium or the content of nutrients in the medium, or to increase the oxygen partial pressure in the fermentation tank. However, these methods have major limitations. Because the composition and temperature of fermentation medium are determined according to the physiological characteristics of known strains and the needs of bioanabolism, they cannot be changed at will. Although the increase of tank pressure increases the oxygen partial pressure, thereby increasing the solubility of oxygen, it also increases the partial pressure of other gases, such as carbon dioxide, and the solubility of carbon dioxide is much higher than that of oxygen, which is not conducive to the fermentation.

  The amount of oxygen can be increased by increasing the amount of ventilation in the fermentation tank, but the amount of dissolved oxygen has a certain upper limit at a certain temperature and pressure. Excessive increase of ventilation will result in a large amount of gas not dispersed, directly through the mixer in the form of large bubble rise, stirring power will be greatly reduced. It may also increase the burden of air filters and the possibility of contamination.

  The second factor is the uneven distribution of oxygen in the fermentation tank, resulting in the lack of oxygen utilization rate.

  The basic limiting factor of oxygen supply capacity in fermentation liquor is oxygen transfer rate. Oxygen is dissolved in water by air and then transferred to the surface of the cells of the bacteria, where it is eventually used. In this process, oxygen transfer resistance mainly includes gas membrane resistance, liquid membrane resistance, mass transfer resistance of cell membrane, etc., which greatly reduces the utilization rate of cell to oxygen.

  At present, most industrial fermenters are used to solve this problem. In the first place, the air flowing into the fermentation liquid can be dispersed into small bubbles and the condensation of small bubbles can be prevented, thus increasing the contact area of the gas liquid. At the same time, it can reduce the bacteria's nodule and the resistance of the liquid membrane around the cells, which is conducive to the oxygen absorption of the bacteria.

  However, the increase of stirring power is limited to a certain extent. Excessive stirring will produce great shear force, resulting in inactivation of microbial species and products. In addition, too much stirring power will also lead to uneconomical system operation and waste of resources.

  Therefore，a new method to increase the low-oxygen condition tolerant ability of the engineering bacteria is of great importance.

  So, we thought that if we made the bacteria more oxygen-carrying, it would improve the method for solving the problem.

  For this purpose，we have found a kind of hemoglobin in Vitreoscilla, which can enhance the ability of oxygen-carrying.

  Hemoglobin is more than just the red pigment in vertebrate blood. In fact, it exists in almost all living organisms, including vertebrates, invertebrates, higher plants, algae, fungi and bacteria. Vitreoscilla is a kind of obligate aerobic gram-negative bacteria living in an environment that lacks of oxygen, such as rotting plant material, cow dung, flooded rice paddies and stagnant ponds. In the 1970s, hemoglobin (VHb) with oxygen binding property was found in Vitreoscilla. Its structure, spectral property, kinetic property of oxygen binding and transport of oxygen were all homologous and similar to the hemoglobin of eukaryotes. At present, the structure and function of the hemoglobin (VHb) of Vitreoscilla has been studied clearly. It is a kind of oxygen-regulated and oxygen-bound protein with strong oxygen binding capacity and can reduce the oxygen demand of bacteria. Under the condition of poor oxygen, Vitreoscilla hemoglobin can improve the cells from the molecular level of dissolved oxygen utilization ability, and can improve the cell oxygen transfer efficiency, satisfy the requirement of the cell respiration, make it adapt to the low dissolved oxygen levels, so as to promote the growth of cells and some metabolites synthesis, the cell alive in the lean oxygen environment, and can improve the yield and yield of desired products in the cell. Therefore, it has a good application prospect in genetic engineering and fermentation engineering. Currently, the gene (VGB) that encodes VHb has been cloned and sequenced, and is expressed in multiple heterologous hosts. Studies have shown that heterologous expression of VHb can promote aerobic growth of escherichia coli lacking terminal oxidase.^[1]

  During aerobic respiration, hypophysis hemoglobin is present in the cytoplasm and peripheral cytoplasm Spaces in the form of biologically active oxygenation.

  However，the cell membrane imposes limits on the transfer of oxygen to intracellular VHb, thus making efficient hemoglobin-oxygen contact a challenge. Based on this, our team designed a VHb surface display system to express VHb on the outermost shell of the bacteria to raise the hemoglobin-oxygen contacting efficiency.

  The cytoderm of gram-negative bacteria consists of a thin layer of peptidoglycan and an outer layer covering it. The outer wall is a membranous structure composed of lipids (including lipopolysaccharides and phospholipids) and characteristic proteins, so it is also called the outer membrane. The cytoplasmic membrane, or membrane, is often called the inner membrane.^[2]

  Our team chose three surface display systems, they are： INP system, intimin system and autotranspoter system.

INP surface display system

  INP is a secretory outer membrane protein, which exists in pseudomoma syringae and pseudomoma fluorescein (P. Flurorescens) and Erwinia sp. In gram-negative bacteria. Its protein-coding sequences are composed of three typical domains: n-terminal domain, c-terminal domain and intermediate repetitive structural units. Among them, the N terminal structure domain (accounts for about 15% sequence) contains abundant aspartic acid and hydrophilic type of serine and threonine, through glycosyl phosphatidyl inositol (GPI) glucosylphosphatidylinositol, anchor is due to the cell surface], is advantageous to the macromolecular surface display of foreign proteins. Studies have shown that INP protein C - domain and internal repeating the lack of domains will not affect the INP outside anchor, transmembrane transport and membrane in INP display system is reported, the N end of INP plus a small amount of repeat unit, INP middle sequence, NC unit combination and separate N terminal structure domain can be used as effective anchoring base sequence in a variety of gram-negative bacteria display system.

intimin surface display system

  Intimin is a virulence factor (adhesin) of EPEC and EHEC E. coli strains, which is expressed on the bacterial cell surface where it can bind to its receptor Tir (Translocated intimin receptor).^[4] Intimin are integrated into the bacterial outer membrane with the amino-terminal region, while the carboxy-terminal region of the polypeptide is surface exposed.^[5]So, protein fused to the C-terminal of intimin can be display on the OM of the bacteria.

autotranspoter surface display system

  Autotranspoter refers to a family of proteins that carry sufficient information on a single gene to direct their own secretion out of a bacterial cell. ATs can insert into the OM(outer membrane) to translocate a passenger domain through the OM, so ATs can form an efficient surface display system because the native passenger domain can be easily replaced by the protein of interest. The first AT to be described was IgA protease of Neisseria gonorrhoeae, and the IgA protease C- terminal domain(Igaβ) has been use successfully for the display of different foreign proteins.^[6]

The Reference

[1]Haifeng Zhao.Structural and functional studies on Vitreoscilla hemoglobin(2011)

[2]Zhaohui Xu.Establishment and applications of a cell surface display system in Escherichia coli(2000)

[3]Mingya LI,Chenshui Lin.Ice crystal nucleoprotein and its application in bacteria surface display technology. Amino acids and biological resources (2016) 38(2) : 7-11

[4]Wikipedia

[5] Thorsten M. Adams,et al. Intimin-Mediated Export of Passenger Proteins Requires Maintenanceof a Translocation-Competent Conformation.OURNAL OF BACTERIOLOGY(2005) p. 522–533

[6] Edwin van Bloois, et al. Decorating microbes: surface display of proteins on Escherichia coli.Trends in Biotechnology(2011), Vol. 29, No. 2