Difference between revisions of "Part:BBa K4719024"
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Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. <br>We employed two strategies to produce this material: <br>
 
Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. <br>We employed two strategies to produce this material: <br>
 
<b>1.</b>The first approach was to add N-acetylglucosamine into the growth medium
 
<b>1.</b>The first approach was to add N-acetylglucosamine into the growth medium

Revision as of 13:49, 9 October 2023


ClCDA chitin deacetylase
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a of bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.

Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer.
We employed two strategies to produce this material:
1.The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013.
2.The second one was the production of N-acetylglucosamine by K. xylinus from common sugars such as glucose, fructose, and sucrose in the growth medium BBa_K4719014.
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer BBa_K4719019, BBa_K4719020, BBa_K4719024.

Usage and Biology

ClCDA is chitin deacetylase isolated from fungus Colletotrichum lindemuthianum. It catalyzes hydrolysis of N-acetamido groups in polymers containing N-acetyl-D-glucosamine monomers. ClCDA requires Co2+ for its catalytical activity.

ClCDA gene consists of two exons and encodes 248 amino acids including extracellular localization signal peptide. Coding sequence excluding signal peptide was cloned into pMAL-p5x-CL-StrepII vector containing MBP (maltose binding protein) sequence in N-terminal and Strep-tag II in C-terminal.

ClCDA can catalyse both deacetylation and acylation reactions by changing reaction conditions [1].

Experimental characterization

Protein expression optimization

ClCDA fused with MBP (72.3 kDa) biosynthesis optimization in ArcticExpress (DE3) E. coli strain. Target protein expression was induced with 0.05 or 0.1 mM IPTG when cell culture optic density at 600 nm reached 0.4 or 0.8. After induction, cultures were incubated at 16 or 28 °C overnight. ClCDA-MBP, expressed in E. coli ER2508, was used as control. The optimal biosynthesis conditions were using ArcticExpress (DE3) E. coli when protein expression is induced at 0.8 OD600 with 0.05 mM IPTG and cultures are incubated overnight at 28 °C after induction.

Figure 1.M - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), S – soluble protein fraction, I – insoluble protein fraction.

Western blot analysis

Western blot for evaluating Strep-tagged ClCDA fused with MBP (72.3 kDa) biosynthesis in ArcticExpress (DE3) E. coli strain. Target protein expression was induced with 0.05 or 0.1 mM IPTG when cell culture optic density at 600 nm reached 0.4 or 0.8. After induction, cultures were incubated at 16 or 28 °C overnight.

Figure 2.M - PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific), S – soluble protein fraction, I – insoluble protein fraction, ctrl – unrelated purified Strep-tagged protein (83 kDa).

Protein purification

Protein purification was performed with 6 grams of biomass using ÄKTA avant chromatography system.
Figure 3.Chromatogram of ClCDA-MBP expressed in ArticExpress (DE3) E. coli strain purification using ÄKTA avant chromatography system. Elution stage peak indicates that new expression conditions are optimal for ClCDA-MBP.

Deacetylation enzymatic activity analysis with fluorescence microscopy

Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase ClCDA - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCL buffer. The samples were incubated for 14 h at 37° while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98°C.

For cellulose-chitosan copolymer generation from cellulose-chitin exopolymer we used chitin deacetylase ClCDA. To determine if the deacetylation of our cellulose-chitin copolymer was successful, we used Alexa Fluor™ 405 NHS ester dye that specifically binds to free amino groups. On that account, only deacetylated copolymers should produce any fluorescent signal at this wavelength. To verify that our purified deacetylases are enzymatically active, at first, we checked the deacetylation activity on enzyme's natural substrate - chitin.

Figure 4:A - K. xylinus modified with AGM1-NAG5-UAP1 BBa_K4719013 producing bacterial cellulose-chitin composite grown on 1% glucose and 1% N-acetylglucosamine. B - K. xylinus modified with AGM1-GFA1-GNA1-UAP1 BBa_K4719014 producing bacterial cellulose-chitin composite grown on 2% sucrose. C - K. xylinus modified with AGM1-GFA1-GNA1-UAP1 BBa_K4719014 producing bacterial cellulose-chitin composite grown on 2% fructose. D - chitin control. ClCDA was active on native substrate chitin and on the bacterial cellulose-chitin copolymer.

Acetylation enzymatic activity analysis with fluorescence microscopy

Acetylation activity was investigated by performing click chemistry reaction on ClCDA acetylated bacterial cellulose-chitosan polymer achieved by deacetylation accomplished by using all of our recombinant deacetylases. Acetylation was performed in 200 µL propiolic acid buffer pH 6.5 (Tris 0.1M, propiolic acid 0.5M) with 2µM ClCDA. The reaction was incubated for 24h at 37°C while shaking.

Click chemistry reaction was performed on propiolated bacterial cellulose-chitosan copolymer after washing 2 times with 0.1M Tris-NaOH pH 6.5 buffer. Adding copolymer to 100 µL HCl (pH 4) and 2.3 µL 3-azido-7-hydroxyazidocoumarin, 23.5 µL sodium ascorbate and 23.5 µL CuSO4 solution, incubated for 10 min. We evaluated the fluorescence (using DAPI light cube) of our copolymer after different wash times (30min and 90min).

Figure 5:A,E - K. xylinus modified with AGM1-GFA1-GNA1-UAP1 BBa_K4719014 producing bacterial cellulose-chitin composite grown on 2% sucrose and deacetylated with ClCDA BBa_K4719024. B,F - K. xylinus modified with AGM1-NAG5-UAP1 BBa_K4719013 producing bacterial cellulose-chitin composite grown on 1% glucose and 1% N-acetylglucosamine, deacetylated with CBD-FRF-ArCE4A BBa_K4719020. C,G - control of bacterial cellulose produced by unmodified K. xylinus grown on 2% glucose. D,H - control of K. xylinus modified with AGM1-NAG5-UAP1 BBa_K4719013 producing bacterial cellulose-chitin composite grown on 1% glucose and 1% N-acetylglucosamine, not deacetylated. A,B,C,D - polymers washed for 30 min; E,F,G,H - polymers washed for 90 min. The control groups has shown a significant loss of fluorescence, thus validating the success of the “click” and therefore the acetylation reaction by ClCDA.

References

1.Linhorst, M. et al. (2021) 'Chitin Deacetylase as a Biocatalyst for the Selective N-Acylation of Chitosan Oligo- and Polymers', ACS Catalysis 11 (23), 14456-14466. doi: 10.1021/acscatal.1c04472