Team:FAFU-CHINA/Description

Team:FAFU-CHINA - 2020.igem.org

Description

Lignocellulose

Plant cell wall is mainly composed of cellulose, hemicellulose and lignin (Tadesse and Luque, 2011). We call these three types of organic polymers collectively referred to as lignocellulose.

The main components of lignocellulose cross-linked to form a dense natural cell wall structure. This complex and stable three-dimensional structure constitutes a natural anti degradation barrier. Therefore, it is difficult for lignocellulose to be directly degraded by enzymes or microorganisms.[1]

How to use cellulose

If we want to use cellulose, we have to remove the lignin and hemicellulose wrapped in it. This is a highly ordered reaction process, which not only requires a large number of enzymes, but also has strict requirements on the time and space of enzyme action. We divided the reaction process into three technical links: pretreatment、cellulose degradation and microbial fermentation (Taherzadeh and Karimi, 2007). Practice has proved that the utilization efficiency of lignocellulose without any pretreatment is generally low. Therefore, pretreatment is considered to be the most important step in the degradation and transformation of lignocellulose (Alvira et al., 2010; Chandra et al., 2007; Mosier et al., 2005).

In lignocellulose, the degradation difficulty of lignin is the highest, followed by cellulose and hemicellulose.[2] The complex spatial structure of lignin not only hinders the enzymatic degradation of cellulose and hemicellulose in the raw materials of lignocellulose, but also produces non productive adsorption on hydrolase, which seriously affects the enzymatic hydrolysis efficiency of substrate (Sasaki et al., 1979). Therefore, we hope that lignin degradation can be separated from cellulose and hemicellulose degradation. By means of inducing expression, we can achieve this goal.

The degradation of lignin —— Fenton reaction principle

Lignin is an amorphous compound, so there is not a set of enzymes that can completely match with lignin degradation. We can only use the Fenton reaction principle to carry on the nonspecific degradation[3,4]. According to the principle of Fenton reaction, we found two enzymes. One is aryl alcohol oxidase (AAO, o94219, from Pleurotus eryngii), which can produce hydrogen peroxide; the other is horseradish peroxidase (HRP, q42578, from Arabidopsis), which can consume hydrogen peroxide and generate free radicals to attack lignin[5,6].

The degradation of hemicellulose

After the limit of lignin degradation is removed, only hemicellulose is left. Hemicellulose is a heterogeneous polymer composed of several different types of monosaccharides, including xylose, arabinose and galactose. The main components of JUNCAO are xylose, glucose and a small amount of arabinose, with trace or no mannose and galactose[7].The results show that the hemicellulose component of JUNCAO is relatively single. Through the joint action of the following six enzymes, we can well degrade the hemicellulose component in the JUNCAO[8].

  1. xynA,Q8GJ44(Endo 1,4 - β - xylanase ，derived from thermophilic carboxybacteria)
  2. xynB,P40942(Xylosidase, derived from thermophilic carboxybacteria)
  3. abfA, α - l-arabinofuranase
  4. aguA, α - glucuronidase
  5. axe2, acetyl xylan esterase
  6. faeA, ferulic acid esterase A

(Due to our limited time and money, we intend to compress the enzyme combination for hemicellulose degradation, and only express xynA and xynB, the core enzyme of hemicellulose, in the system, because our main purpose is to remove the restriction of hemicellulose on cellulose degradation, not to completely degrade hemicellulose.)

The degradation of cellulose

The last is the degradation of cellulose: the most classical cellulose degradation model considers that cellulose is divided into crystalline and non-crystalline regions in structure. The cellulose chains in the crystalline zone are closely arranged without voids, while the cellulose chains in the amorphous zone are loosely arranged with large pores. Under the guidance of cellulose binding domain (CBM), endocellulase can enter the amorphous region, cut off the cellulose chain from the inside, and then expose more cellulose chain end sites for exocellulase. Under the action of exocellulase, the cellulose chain is shortened and cellobiose is produced. Finally, cellobiose is decomposed into glucose by β - glucosidase, which completes the degradation process of cellulose[9].

Fig 4. Cellulose degradation model The picture on the left shows the binding of the two enzymes to the cellulose chain before degradation, the picture on right shows the degradation of crystalline and non-crystalline regions and the degradation of cellobiose by β – glucosidase.

In fact, the crystalline region of cellulose can also be degraded. Therefore, we found an enzyme combination, LPMO (a3kkc4), which is similar to Fenton reaction and can act on the degradation of lignin and cellulose at the same time. And its coenzyme, CDH (q01738, cellobiose dehydrogenase) [10].

Fig 5. The combination of LPMO and CDH act on the degradation of lignin and cellulose

How can LPMO degrade cellulose and lignin in dense crystalline zone? The answer lies in its spatial structure. LPMO is shaped like a triangular cone, and the active center of LPMO is located on its flat bottom surface. This structure makes LPMO work closely on the surface of the substrate.

Fig 6. Artist impression of the interaction between CBP21 and chitin (side view, left top view, right). The picture highlights how the fiat surface of CBP2i1 fits the flat surface of a β-chitin crystal (the binding interaction is hypothetical and has not been modelled). the surfaces of residues known to interact with chitin [72] are coloured magenta and the side chains of these residues are shown. in the side view some of the magenta surface is hidden by the white surface of other residues. Please note that the actual orientation of the enzyme relative to the substrate is unknown; see[18]for an interesting discussion of this topic.

The sources of lignocellulose

There are many sources of lignocellulose [11]. We prefer to use prokaryotic enzymes. On the one hand, the lignocellulose from prokaryotes has good heat resistance (such as Clostridium fibrinolyticum, Clostridium thermophilus, Bacillus thermothermothermophila, Clostridium thermophilus); on the other hand, the lignocellulose from prokaryotic cells contains a natural dockerin domain, which can be adapted to be added into the fibrous bodies (such as Clostridium cellulolyticum).

Fibrosome

Fibrosome is a kind of multi enzyme complex structure formed by many kinds of cellulase and hemicellulase by anchoring adhesion mechanism[12]. Fibrosomes are attached to bacterial cell walls by cell adhesion proteins, which can degrade natural cellulose materials efficiently and thoroughly.

Fig 7. Superposition of BcCohl with the type l cohesin-dockerin complexes from C.thermocellum. Structural overlay of C.thermocllun type cohesin-dockerin complexes in two binding orientations(PDB codes 1ohz and 2ccl) superposed with BcCoh. Residues involved in C. thermocelum cohesin-dockerin binding are highlighted in stick representation. Dockerin residue numbers are boxed and indicate equivalent residue positions on CtCoh-1ohz (gray colored structure) and CtCoh-2ccl (brown color) respectively. The cohesin residue numbers refer to structurally equivalent positions on BcCohl (green)and CtCoh-1ohz-A(salmon), respectively. Calcium ions are represented as spheres colored in purple. The top inset is a magnified view from the opposite side, around the blue spherical surface that highlights the canonical C.thermocllum Ser/Thr interface residue pair. Figures were prepared using UCSF Chimera [38]. Fig 8. The method to construct artificial fiber bodies

Artificial fiber bodies

It is very difficult to heterologous expression of bacterial natural fibrosomes in chassis organisms, because the molecular weight of fibrosomes is very large and contains a large number of repetitive sequences. By consulting the relevant literature, we found a method to construct artificial fiber bodies. It includes recombinant enzyme with docking protein domain, secondary scaffold protein with adhesin domain and scaffold protein with lectin subunit[13].

An integrated system

Combined bioprocessing is a more economical and convenient process in the production of biofuels, which integrates cellulose secretion, cellulose hydrolysis and ethanol fermentation into one step and directly uses cellulose micro carbon source to produce ethanol. The fermentation system needs to complete the following four tasks: lignin degradation, hemicellulose degradation, cellulose degradation, ethanol fermentation. Therefore, we need to design an integrated system to connect these four steps closely without interfering with each other. Therefore, our idea is to design a co-culture system composed of three kinds of chassis organisms. The initial concentration of biomass and biomass can be adjusted dynamically by the initial concentration of biomass.

Fig 9. The selection of chassis organisms

About the selection of the system, we have come up with two feasible system designs: bistable system and double plasmid system. As mentioned above, the complex spatial structure of lignin not only hinders the enzymatic degradation of cellulose and hemicellulose in lignocellulosic materials, but also produces non productive adsorption of hydrolases, which seriously affects the enzymatic hydrolysis efficiency of substrates. Therefore, we hope that the degradation of lignin can be separated from the degradation of cellulose and hemicellulose. We can achieve this goal by inducing expression[1].

And the bistable system can switch gene routes between two different stable states by artificial addition of inducers.

Fig 10. double plasmid system

However, its disadvantages are: (1) it is rather cumbersome to add inducer into the fermentation tank at the right time; (2) the inducer used is expensive and has certain toxicity to human and chassis organisms; (3) the use of inducer will introduce impurities into the system. Two plasmid system: Lactose induced expression plasmid and lactose induced non expression plasmid

Fig 11. Lactose induced expression plasmid Fig 12. lactose induced non expression plasmid

A certain amount of lactose was added into the fermentor to induce the expression or non expression of the plasmid. The advantages are as follows: (1) there is no need to find the right time to release inducer in the fermentation process; (2) taking the inducer IPTG used in the bistable state as an example: IPTG is expensive and can not be biologically utilized by the chassis; lactose, as an inducer, is not only cheap, but also can be used as a transitional carbon source by the chassis.

Through two-phase comparison, we finally chose the double plasmid system. The final desired effect is that when lactose exists, it can induce the expression of lignin enzyme and inhibit the expression of cellulose and hemicellulase; when lactose is exhausted, the expression of lignin enzyme stops and the expression inhibition of cellulase and hemicellulase is relieved.

The selection of chassis organisms

Selection of chassis organisms: yeast and Escherichia coli are facultative anaerobic organisms, one is the common chassis in eukaryotes, the other is the common chassis in prokaryotes. Because the integrated fermentation system needs to design additional control system, we chose the more simple Escherichia coli as the core chassis of the whole system to undertake various degradation functions, and let the yeast undertake the functions of ethanol fermentation, scaffold protein production and auxiliary degradation of lignocellulose. Cellulase and hemicellulase used,

After consulting UniProt database, we know that the optimum temperature is about 60 ℃ and the optimum pH is about 6-7. Generally speaking, the culture temperature of E.coli is 37 ℃, under this temperature, the lignocellulose enzyme can secrete normally, but its degradation ability is low. Relevant literature points out that under the condition of constant pH and temperature of 40 ℃, the enzyme activity of lignocellulose can still be maintained at about 95% after 24 hours. So our idea is to first let chassis 2 secrete enough lignocellulose enzyme, and then raise the temperature of fermentation tank to 50-60 ℃. Generally speaking, the fermentation temperature of Saccharomyces cerevisiae is about 30 degrees, which obviously does not meet the requirements of the system. Therefore, we chose Kluyveromyces Maximus, which can normally perform the fermentation function at about 50 degrees, as the chassis III. In addition, Kluyveromyces maximus also has the ability to utilize xylose, which means that it can convert xylose, the degradation product of hemicellulose of JUNCAO, into ethanol.

Fig 13. Metabolic pathway of xylose degradation by Kluyveromyces Maximus Work flow of integrated fermentation system In the system, the first chassis is Kluyveromyces Maximus, which contains lactose operon plasmid. The second plate was E•coli (BL21), a non-gate plasmid containing lactose operon. The third chassis is Kluyveromyces Maximus, which contains the scaffold protein genome integration plasmid.

Work flow of integrated fermentation system

In the system, the first chassis is Kluyveromyces Maximus, which contains lactose operon plasmid. The second plate was E•coli (BL21), a non-gate plasmid containing lactose operon. The third chassis is Kluyveromyces Maximus, which contains the scaffold protein genome integration plasmid.

Fig 14. Work flow of integrated fermentation system

Firstly, a certain amount of lactose is added into the fermentation tank. At this time, chassis 1, chassis 2 and chassis 3 start to consume lactose for proliferation. Due to the presence of lactose, chassis II cannot express cellulosic and hemicellulose degrading enzymes, while chassis I can secrete enzymes that degrade lignin. Of course, chassis III is not idle. During the whole process of lignocellulose degradation, it expresses enzymes that can act on both lignin degradation and cellulose degradation. With the consumption of lactose, the expression of cellulase and hemicellulase in chassis 1 was stopped due to the lack of inducer; the lactose non gate in chassis 2 also failed, so cellulose and hemicellulase were expressed. It can be seen that the process of lignin degradation and cellulose degradation has been effectively separated due to the double plasmid system. With the passage of time, chassis 2 and chassis 3 began to accumulate lignocellulose. This is because the optimal temperature of the selected lignocellulose is about 60 ℃, so the degradation ability of lignocellulose is limited before the temperature of fermentation tank is raised, so they will retain the active form and accumulate in the system. When the accumulation reaches a certain level, we can raise the temperature of the fermentation tank and formally degrade cellulose and hemicellulose. Since it has accumulated enough lignocellulose, chassis II has accomplished all its tasks with glory, and it soon dies because it can't tolerate the high temperature. In the process of the accumulation of lignocellulose, chassis 3 also expressed the scaffold protein and combined the lignocellulose produced by chassis 2 on the cell wall of chassis 3 in the way of self-loading to form large cellulosic bodies. Cellulosome not only has a more efficient degradation rate than free enzymes, but also can enrich a large amount of glucose in yeast cells, which is very beneficial to the production of ethanol from glucose in chassis three and chassis one. Finally, the reaction was carried out to the stage of ethanol fermentation. Kluyveromyces maximus used soluble reducing sugar to produce ethanol through anaerobic respiration. Because the ability of yeast to tolerate ethanol is limited, the concentration of ethanol in the system will reach a stable level after the fermentation lasts for a period of time. At the end of the integrated fermentation process, we will further increase the temperature of the fermentation tank. On the one hand, it is to concentrate the mother liquor of ethanol; on the other hand, it is to use high temperature to kill the chassis organisms used in the fermentation system to avoid potential safety problems.

Integrating industrialization ideas into our project design

We rely on the chassis to produce enzymes, which can be directly secreted into the fermentation tank, thus avoiding cumbersome intermediate processes (such as breaking cells). Since we do not add enzymes directly to the system, we can reduce or completely eliminate the cost of enzyme extraction, purification, preservation and transportation compared with traditional biological industry. The chassis selected is highly biosafety and well researched, and has been widely used in industrial production. We are committed to the design of high temperature fermentation system, because high temperature conditions can improve the reaction speed and avoid contamination of the system by miscellaneous bacteria (which can reduce the cost of using antibiotics and preservatives).

Reference:

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