BJRS

Background

The importance of industrial biotechnology increases with time

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 of China issued the “The 13th five-year plan for biological industry development”, which proposed that by 2020, the output value of bio-manufacturing industry will exceed one trillion RMB in 2020, and the proportion of bio-based products in all chemical production will reach 25%, and the annual replacement of fossil energy by biological energy will exceed 56 million tons of standard coal, achieving the goal of comprehensive application in areas such as electricity, gas supply, heating, fuel.

Poor oxygen usage of engineering bacteria limits the development of industrial biotechnology

In spite of the high attention, the commercialization of industrial biotechnology is not as fast as 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 leverage the technology of high-cell-density growth, and the key factor limiting cell density in bioreactors is the availability of oxygen during the late phases of fermentation^[1].

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.

Factors that affect the oxygen content in the fermentation process.

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

1. The 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 ℃, 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].

2. The uneven distribution of oxygen in the fermentation tank, which is mainly affected by the 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 transfers 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.

The limitations and recent methods to increase the oxygen usage in the fermentation process

1. 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 many limitations. The temperature and nutrients of fermentation medium are determined according to the physiological characteristics of the stains, so 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.

2. Increasing the amount of ventilation in the fermentation tank.However, 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. Morever, 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.

Vitreoscilla hemoglobin(VHb) are well used to the oxygen usage ability of bacteria.

Hemoglobin is not only existed in vertebrate blood but appears 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 VHb 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, VHb can improve the dissolved oxygen utilization ability of bacteria, and can improve the cell oxygen transfer efficiency. Therefore, the expression of VHb can satisfy the requirement of respiration and help the bacteria adapt to the low dissolved oxygen levels, thus can promote the growth and metabolism of cells, improving the yield of desired products. 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 has been expressed in multiple kinds of hosts. Studies have shown that heterologous expression of VHb can promote aerobic growth of E.coli^[8].

Proposed mechanism of VHb interaction with cytochrome complex to promote energy metabolism.(L. Zhang *et al.*, Biotechnology Advances, 2007)

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.

Working model for INP drived surface display

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.

Working model for intimin driven surface display

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]

Working model for autotransporter drived surface display

References

[1]Guoqiang Chen. New challenges and opportunities for industrial biotechnology. Microb Cell Fact.11(2012)

[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 38(2) : 7-11(2016)

[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

[7] Lei Zhang et al.. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnology Advances 25(2007).