Part 1 Construction of engineered strains

1.1 The construction of recombinant plasmids for high expression of enzymes

Using the principle of homologous recombination, the primers were designed to construct recombinant plasmids. In phka plasmid, the promoter and signal peptide were changed, and AOX1 was changed into AOXm or GAP, and α -signal peptide was changed into MF peptide.

Figure 1 Figure 1 Recombinant Plasmid map

PCR to obtain the gene of interest and vector fragments

1:AOXm-IsPETase^PA skeleton(8240bp)；2:MF-IsPETase^PA skeleton (7628bp)

Figure 2 Identification of AOXm-IsPETase^PA and MF-IsPETase^PA promoter fragment from PCR or enzyme digestion

Table 1 The concentration of elements for plasmids construction

Element	ng/µL	A260/280	A260/230
AOXm-IsPETasePA skeleton	38.9	1.84	1.42
MF-IsPETasePA skeleton	167.9	1.81	1.31

Figure 3 Identification of AOX1 promoter,α-signal peptide and GAP promoter fragment from PCR or enzyme digestion

Table 2 The concentration of elements for plasmids construction

Element	ng/µL	A260/280	A260/230
α-signal peptide	128.8	1.89	0.21
GAP	207.3	1.86	0.64
AOX1	120	1.85	1.15

We introduced plasmids into E. coli and performed colony PCR after colonies were grown, with bands of 267 bp, 944 bp and 477 bp for α-signal peptide, AOX1 signal peptide and GAP signal peptide, respectively

Figure 4 Identification of α-signal peptide, AOX1 signal peptide , GAP signal peptide promoter fragment from PCR or enzyme digestion

Conclusion: The correctness of each plasmid element was verified by PCR and electrophoresis, and we successfully obtained the desired plasmid element.

Figure 5 Sequencing map

1.2 Construction of plasmids for enzyme mutagenesis

Figure 6 Different expression elements

1. 29 Mutation site, 2. 59 Mutation site, 3. 122 Mutation site, 4. 183 Mutation site
Figure 7
Table 3 The concentration of plasmids

Index	ng/µL	A260/280	A260/230
29	91.4	1.82	1.95
59	68.9	1.80	0.52
122	50.4	1.86	0.64
183	70.8	1.82	0.67

Results: The above seven plasmids were successfully constructed and introduced into E. Coil, and they were verified by sequencing. 92,169,190,212,223 five plasmids were successfully constructed and introduced into Escherichia coli, and the sequencing was successful. We then linearized the plasmid and ran the glue to verify whether the band was single.

Figure 8
Table 4

Index	ng/µL	A260/280	A260/230
212	41.9	1.74	0.56
223	15.5	2.00	1.70
190	43.9	1.64	0.68
169	13.9	1.80	0.65
92	39.4	1.68	0.88

Then we performed PCR product purification and electrotransformation, and transferred the plasmid into the yeast, and then we performed the yeast colony PCR, and the rubber was successfully verified.

Figure 9 Colony PCR at glycosylation site

1.3 The construction of recombinant strains and protein expression identification

Three recombinant plasmids (AOX1-MF-IsPETase^PA, AOXm-α-signal peptide-IsPETase^PA, GAP-MF-IsPETase^PA, GAP-MF- IsPETase^PA) and four mutant plasmids of IsPETase^PA (29,59,122, 183 glycosylation sites) were constructed and introduced into Pichia pastoris.

Plasmid linearization

After crushing and extracting the plasmid, we sent the samples for sequencing, all of which were successfully verified. Then, we linearized the plasmid.

4. 29 Mutation site, 5. 59 Mutation site, 6. 122 Mutation site, 7.183 Mutation site
Figure 10 Colony PCR at glycosylation site No. 29-183

Figure 11 Colony PCR at glycosylation site

Figure 12 Colony PCR at AOXm-α-signal peptide-IsPETasePA, GAP-MF-IsPETasePA, AOX1-MF-IsPETasePA

However, the colony on the AOX1 plate was transparent instead of white, so we suspected that the plasmid did not successfully introduce Pichia pastoris, but was still in Escherichia coli, so we re-switched it and ran the glue to verify.

Figure 13 Linearized plasmid map

Figure 14 AOX1 colony PCR

The biomass and protein level of recombinant strains

Figure 15
1：AOXm-α-signal peptide-IsPETase^PA，2：GAP-MF-IsPETase^PA；
3：AOX1-MF-IsPETase^PA；4：AOXm-MF-signal peptide-IsPETase^PA

Figure 16
1: IsPETase^PA; 2: Deglycosylation of IsPETase^PA; 3: 29 mutant IsPETase^PA;
4: 59 mutant IsPETase^PA;5: 122 mutant IsPETase^PA; 6: 183 mutant IsPETase^PA

Figure 17

Figure 18 29,59,122,183 mutant IsPETase^PA

Part 2 Verification of plastic degradation conditions

Figure 1

Figure 2

In a 96-well plate at 40°C, we found that glycosylation mutation site 122 was more efficient than the other mutation sites, and that glycosylation mutation site 122 was more efficient than other pH conditions at pH 9.0 and 10.0. Secondly, we found that at pH 9.0, we found that glycosylation mutation site 122 could significantly degrade PET at 30°C, 40°C, and 50°C, which showed that our mutation could improve the thermal stability of the enzyme. Thirdly, at 30°C, the glycosylation mutation site No. 122 could degrade plastics with high efficiency at pH 6.0 and 7.0, but the enzymatic reaction conditions of IsPETase^PA in the original literature were alkaline, and the optimal pH was 9.0, so the mutation could broaden the acid-base tolerance of IsPETase^PA and make it degrade under neutral conditions.

Part 3 Optimization of culture conditions for mixed fermentation

3.1 Investigation static and dynamic condition for mixd-fermentation

The ration of GS115/mRFP to DSM 2004 was 1:1 at OD (both 0.05). Dynamic and static cultures were carried out separately in different medium including HS, HS-Sucrose, BMMY, YPS liquid medium with different pH such as 6.0 and 7.0.

Figure 1

Figure 1 show that the number of DSM 2004 is little or none under dynamic culture conditions, while the yeast shows good growth. At the same time, we observed that the production of bacterial cellulose was inhibited. This indicated that the growth and production of DSM 2004 were affected in the dynamic mixed culture mode. We need to explore new models of training.

In conclusion, dynamic culture is more suitable for yeast growth, and static culture is more suitable for Komagataeibacter xylinus growth.

3.2 Optimization of medium component for mixd-fermentation

Mixed fermentation with different nitrogen sources or carbon sources

We used different kinds of media, such as HS-Sucrose, BMMY, HS, pH set to 6.0, 7.0.In order to make the BC film-formed, we chose a static culture method, and the BC membrane was extracted after 5 days of culture.

First, we filter and collect the BCs and rinse repeatedly with deionized water to remove the medium. BC was immersed in 0.2mol/L NaOH solution in a water bath at 70~80 ℃ for 2h to remove the residue, and the pH value was about 7.0 by rinsing with deionized water for many times, and finally a milky white translucent BC gel liquid film could be obtained, which was then placed at room temperature for drying for 12h and weighed，thus deriving the yield of BC

More bacterial cellulose in YPS than in HS-Sucrose medium at the same pH.

Figure 2

Figure 3
Table 1 The weigh of BC in different mediums (dry weight)

Medium	pH	Dry Weight
HS-Sucrose	6	0.0713 g
HS-Sucrose	7	0.0907 g
YPS	6	0.1083 g
YPS	7	0.1142 g
BMMY	6	/
BMMY	7	/

Results: The BC yield of YPS medium was higher than that of HS-Sucrose, and the yield of YPS medium was higher than that of pH 6.0 under pH 7.0 .

Figure 4
Table 2 The weigh of BC in different mediums (dry weight)

Medium	pH	Dry Weight
HS-Sucrose	7	0.0153 g
YPS	6	0.0131 g
HS	7	0.0224 g
HS	6	0.0165 g

Yield：HS pH=7＞HS pH=6＞HS-Sucrose pH=7＞YPS pH=6

Results: The BC yield of HS medium was higher than that of HS-Sucrose, and the yield of HS medium was higher than that of pH 6.0 under pH 7.0 .

Figure 5

Results: Through BMMY, YPS and HS analysis, it was found that HS>YPS>BMMY (pH 6.0) was compared in terms of BC yield

Figure 6

Results: Through BMMY, HS and HS-Sucrose analysis, it was found that HS ＞ HS-Sucrose ＞ BMMY (pH 7.0) was compared in terms of BC yield

Figure 7

Results: Through HS(pH6.0 and pH7.0), HS-Sucrose(pH 7.0)and YPS(pH6.0) analysis, it was found that HS (pH7.0)＞ HS (pH6.0), YPS (pH6.0)＞ HS-Sucrose (pH7.0) was compared in terms of BC yield To sum up pH=7 HS ＞ pH=6 HS ＞ pH=6 YPS ＞ pH=7

Table 3 The component of different medium

Medium	Yeast Extract	Tryptone	Glucose	Cane Sugar	Yeast Nitrogen Base	Citric Acid	Disodium Hydrogen Phosphate	Phosphate Buffer
HS	5 g/L	5 g/L	20 g/L			1.15 g/L	2.7 g/L
HS-Sucrose	5 g/L	5 g/L		20 g/L		1.15 g/L	2.7 g/L
BMMY	10 g/L	10 g/L			13.4 g/L		100 mmol/L
YPS	10 g/L	10 g/L		20 g/L

Analysis:

1. By comparing the yield of bacterial cellulose in HS and YPS, it was found that at a lower concentration of yeast extract and tryptone, the yield of HS with glucose as the main carbon source was higher than that with Sucrose as the main carbon source. Therefore, it was believed that carbon source was the main reason affecting the yield of bacterial cellulose. The BC yield in the medium with glucose as the main carbon source is higher than that in the medium with sucrose as the main carbon source, which further proves the conclusion. However, glucose inhibited promoter expression, sucrose promoted the growth of Acetobacter xylosa, and sucrose may not inhibit promoter expression.

2. Through the different inoculation amounts of experiment 1 and experiment 2, it is suspected that OD and bacteria ratio will affect the production of bacteria

3. By means of HS-Sucrose and YPS, under the condition of the same concentration of sucrose, the yeast extract and tryptonose of YPS were higher, and the yield of bacterial cellulose was better, so it was suspected that this was the secondary reason affecting the yield of BC

4. Through co-culture +EG experiment, it was found that PET degradation of EG and TPA was also one of the reasons affecting BC production. Therefore, in the future, single factor optimization experiments will be conducted using BMMY as the base medium

3.3 Optimization of pH for mixd-fermentation

mRFP is a red fluorescent protein that can be used to report protein expression in Pichia pastoris. Choose two different pH conditions (pH 6.0), culture the yeast strain expressing the red fluorescent protein, and measure their cell biomass (OD₆₀₀) and red fluorescence intensity. BMMY culture medium was inoculated with GS115/mRFP at pH 6.0 or 7.0. The red fluorescent protein content and OD value were measured for five consecutive days.

Figure 8 OD₆₀₀ value and red fluorescence intensity of GS115/mRFP

Conclusion: Under different pH conditions, the growth and red fluorescence intensity of GS115/mRFP is similar，which reaches its maximum value at 120 h.

Part 4 Exploration of fermentation conditions

Carbon sources and ratio of DSM 2004

Sucrose and glucose were used as the main carbon sources to carry out fermentation experiments in 96-well plates. The fermentation system (fermentation broth + carbon source solution + bacterial solution) was prepared with 200 μL as the total volume, the initial OD₆₀₀ of DSM 2004 was 0.1. Firsly, we tested the role of sucrose in Bacterial cellulose production. 1g/mL sucrose added to the micro culture system and static fermentation carried out for 7 days. The bacterial cellulose was measured and weighed subsequently (See Figure 1).

Figure 1

Result: Most of the media do not form a bacterial cellulose membrane, and only a small amount of bacterial cellulose membrane appears in the top layer of the liquid, and does not form a white film-like substance. We guessed that it might be because of the carbon source and bacterial load, so we explored the ratio of DSM 2004 bacteria and also replaced sucrose with glucose.

Set the OD gradients to 0.1, 0.5, and 1.0 for fermentation experiments in a 96-well plate. The fermentation system (fermentation broth + glucose solution + medium) was prepared with an OD of 0.1, 0.5 and 1.0 with a total volume of 200 μL, respectively, and the bacterial cellulose was weighed after static mixed culture for seven days. Subsequently, it was found that the yield of OD 0.1 was less, and the yield of 0.5 and 1.0 was more and similar.

The fermentation experiment was carried out in 96-well plate with glucose as the main carbon source. The total volume was 200μL, the DSM 2004 OD was controlled to be 0.5, and the fermentation was carried out according to the fermentation system of 141μL fermentation solution +8μL 0.5g/mL glucose solution +51μL bacterial solution. The fermentation solution was from the medium containing IsPETase^PA , Fast-PETase-212/277, Fast-PETase-212/277-MHETase, IsPETase^PA-MHETase with or without PET degradation products at pH 6.0 or 7.0. All the BC membranes were intact after seven days of culture. (See Figure 2)

Figure 2

The fermentation broth without adding PET and adding PET for five days for degradation was used as the basic system, and the DSM 2004 OD was 0.5 and the glucose solution of the same concentration was controlled, and the fermentation system (fermentation broth + carbon source solution + bacterial liquid) was prepared, and the bacterial cellulose was weighed after standing and mixed culture for seven days.

Figure 3 BC membrane

Figure 4 BC membrane after natural air-drying 1:IsPETase^PA BMMY pH 7.0；2:IsPETase^PA+PET BMMY pH 7.0；3:IsPETase^PA+PET BMMY pH 6.0；4:IsPETase^PA BMMY pH 6.0；5:MHETase-IsPETase^PA BMMY pH 7.0; 6:MHETase-IsPETase^PA+PET BMMY pH 7.0; 7:MHETase-FAST-PETase-212/277 BMMY pH 7.0; 8:MHETase-FAST-PETase-212/277+PET BMMY pH 7.0;
Table 1 Air-dried BC membrane weighing (BMMY pH 7.0)

Sample	Weight
IsPETasePA	0.0017
IsPETasePA + PET	0.0019
MHET-IsPETasePA	0.0021
MHET-IsPETasePA + PET	0.0022
MHET-FAST-PETase-212/277	0.0030
MHET-FAST-PETase-212/277 + PET	0.0035

Table 2 Air-dried BC membrane weighing (BMMY pH 6.0)

Sample	Weight
IsPETasePA	0.0012
IsPETasePA + PET	0.0015

In comparison between the fermentation broth containing enzymes and the fermentation broth containing PET degradation products and enzymes, under the basic conditions of BMMY at pH 7.0, the dry weight of bacterial cellulose for IsPETase^PA+PET increased by 11.76% compared to IsPETase^PA; the dry weight of bacterial cellulose for MHET-IsPETase^PA+PET increased by 4.76% compared to MHET-IsPETase^PA; and the dry weight of bacterial cellulose for MHET-FAST-PETase-212/277+PET increased by 16.67% compared to MHET-FAST-PETase-212/277. Under the basic conditions of BMMY at pH 6.0, the dry weight of bacterial cellulose for IsPETase^PA+PET increased by 25.00% compared to IsPETase^PA.

According to the above data, the degradation products of PET, EG and TPA, can be utilized by DSM 2004 to convert into high-value bacterial cellulose, further proving the feasibility of this project in creating economic effects through product utilization.

Part 5. Results of extended experiments

Dual-enzyme system

1. The construction of M-F and M-Is We took MHETase plasmid as the skeleton, performed enzyme digestion with Bamh I restriction enzyme, linearized the circular plasmid, and then performed enzyme digestion with Bamh I and Bgl II restriction enzyme to obtain FAST-PETase-212/277 and IsPETase^PA fragments. They were then linked with T4 ligase, and finally linearized with BamH I or Bgl II restriction enzymes for banding verification.

2. Fermentation

Figure 1 Protein concentration of M-F and M-Is Black: the construction of M-F in the E.coli; Red: the construction of M-Is in the *E.coli*; Blue: the construction of M-F in the GS115/mRFP; Green: the construction of M-Is in the GS115/mRFP

Figure 2 The OD₆₀₀ of M-F and M-Is

3. Degradation of PET

It was found that under static conditions at 40℃, the IsPETase^PA-MHETase double enzyme system increased by 46% compared with single enzyme, and the FAST-Petase-212/277 double enzyme system increased by 11% compared with protein.

Multiple copy number

1. The construction of IS-2C and IS We successfully constructed a two-copy plasmid for IS-2C.

Figure 3 construction of the IS-2C in the E.coli TOP10

There were two bands and the band length was correct, the plasmid was correct and successful construction, the linearized plasmids were introduced into Pichia Pastoris GS115/mRFP correctly by electrotransfer for expression.

Figure 4 The enzyme digestion results of IS-2C

The band was correct, and a Pichia yeast strain expressing IS-2C was successfully constructed.

2. Fermentation

IS-2C and IS were fermented in BMMY medium to compare the protein concentrations.

Figure 5 Protein concentration of the IS

Figure 6 Protein concentration of IS-2C

By comparison, it is seen that after fermentation has reached the second day, the protein concentration of IS-2C has exceeded the highest protein expression level of IS, and the protein concentration of IS-2C is steadily increasing. It follows that we have successfully increased the expression level of IsPETase^PA by constructing a multi-copy system.

Fusion protein

PETase-mcherry-SZ2 & RGG-SZ1

1. PETase-mcherry-SZ2

Figure 7 *E. coli* expressing the red fluorescence

We have successfully cultured E. coli expressing PETase-mcherry-SZ2.

Figure 8 *E. coli* body precipitation

2. PETase-mcherry-SZ2 and RGG-SZ1

Figure 9. PETase-mcherry-SZ2 and RGG-SZ1 binding

It can be seen that under the microscope, PETase-mcherry-SZ2 expressing red fluorescence condenses with RGG-SZ1, proving that our multi-enzyme agglomerate work successfully and indeed links the two enzymes together.