Characterization

BBa_K3241005: Cel9D

Recombinant plasmid containing CtCel9D gene, BBa_K3241005, was constructed for cellulase expression and transformed into Zymomonas mobilis. Intracellular fraction (crude enzyme solution) were prepared by sonication and cellulase activity was determined using the method of DNS. Details and results are as follows.

Extraction of intracellular crude enzyme solution.

1. Collect 100 mL fermentation broth at 4000 rpm 5 min in 4 °C using 50 mL centrifuge tubes.

2. Use 10 mL PBS to bacteria.

3. 4m/s 40s and suspend 20s, repeat twice in high speed grinding machine.

4. 12000 rpm centrifuge for 10 min in 4 °C.

5. Supernatant is intracellular crude enzyme solution.

Extraction of extracellular crude enzyme solution.

1. Collect 100 mL the supernatant of centrifugation of fermentation broth.

2. Slowly add anhydrous ammonium sulfate powder (48.1 g anhydrous ammonium sulfate per 100 mL bacterial solution) in batches on ice at a precipitation concentration of 80%.

3. Stay overnight in 4 °C.

4. Centrifuge for 13000 rpm 5min. Retain the precipitation. 5. Add 2 mL PBS for per 100 mL fermentation broth. Resuspend and get the extracellular crude enzyme solution.

5. Add 2 mL PBS for per 100 mL fermentation broth. Resuspend and get the extracellular crude enzyme solution.

The DNS method for enzyme activity assay

1. Add 500 L 1 % CMC, 250 L citrate buffer solution (pH=6), 250 L crude enzyme solution.

2. Incubate at 30 °C for an hour.

3. Add 500 L DNS and incubate at 100 °C for 5 min.

4. Detect at 540 nm after cooling and compare with the standard curve.

Measurement of standard curve

Add the reaction system as shown below, incubate at 100 °C for 5 min. Measure the absorbance at 540 nm. Take absorbance as ordinate, glucose concentration as abscissa, make glucose standard curve:

glucose concentration (mg/mL)	0	0.05	0.1	0.15	0.2	0.25	0.3	0.35	0.4
1 mg/mL Glucose solution	0	50	100	150	200	250	300	350	400
ddH20 (mL)	500	450	400	350	300	250	200	150	100
citrate buffer solution (pH=6) (mL)	250
1% CMC (mL)	500
DNS (mL)	500

Fig 1. the activity of the cellulase experimented with the DNS method.

BBa_K3241040:Pgap-PhbC-PhbA-PhbB

This part is a new part, which consists of BBa_K3241000, BBa_K3241022, BBa_K3241112, BBa_K2800032, BBa_K2800013. It is used to construct the PHB biosynthetic pathway in Z. mobilis.

Here, we introduced phbA, phbB, and phbC genes from Ralstonia eutropha to modify the metabolic pathway in Z. mobilis. When Z. mobilis disassembles sugar molecules to produce pyruvate, one part of the metabolic flux directs towards ethanol production catalyzed by pyruvate decarboxylase and ethanol dehydrogenase. Another part pyruvate oxidation decarboxylation to acetyl-CoA. Acetyl coenzyme A translates into PHB under the catalysis of β-ketothiolase, acetoacetyl-CoA Reductase and PHB polymerase.

Fig 2. Key enzymes in PHB synthesizes

Fig 3. Heterogenous PHB biosynthesis pathway

We used the promter Ptet to explore the growth of recombinant strains under the induction of different concentrations of tetracycline. The original can refer to BBa_K3241029.

Fig 4. Orijinal design

Fig 5. Growth Curve

Fig 6. PHB Production

The expressionc of PhbA PhbB and PhbC have little influence on the growth of wild-type Z. mobilis. And we have successfully detected PHB in Z. mobilis by HPLC. But the problem is that the productin of PHB is low. The strong promoter Pgap is selected as the biological element to optimize the metabolic pathway of PHB by increasing the content of precursor acetyl-coA and NADPH. We selected several endogenous genes related to these two influencing factors. fAnd enhanced their expression separately.

Fig 7. Metabolic pathway optimization diagram

For details of the originals, please refer to
BBa_K3241041
BBa_K3241042
BBa_K3241043
BBa_K3241044

Fig 8. Growth curves of different recombinant strains

Fig 9. The production of different recombinant strains

By enhancing the expression of ZMO0367 (BBa_K3241023), ZMO1329 (BBa_K3241024), we successfully increased the production of PHB in Z .mobilis and the maximum production is 7%.

BBa_K3241111:T-1155R

T-1155R is a reverse terminator in Z. mobilis which is found by analyzing omics data. We introduced it into the dual-reporter gene system and measured the intensity ratio of upstream and downstream fluorescent proteins.

Fig 10. The schematic diagram of the dual-reporter gene system

The ratio of GFP to mcherry is 1.107 with T-1155R inserted and 5.433 without terminator.

Termination Efficiency is calculated as 1-1.107/5.443=0.797

Team:HUBU-WUHAN/Characterization