Abstract

Lignocellulosic biomass, the most abundant renewable resources in nature, represents a promising alternative to fossil fuels for sustainable production of chemicals and material. Most of the available lignocellulosic biomass is not a food source for humans and its industrial exploitation does not conflict with agricultural usage. However, the main technological obstacle for industrial exploitation of biomass is the recalcitrant nature of lignocellulosic polymers. One approach to harness the energy contained in biomass is to convert the lignocellulosic polymers in simple sugars, which can be further transformed in valuable compounds, using microorganisms. Some anaerobic microorganisms have developed approaches to break down recalcitrant plant biomass using elaborate extracellular enzyme complexes, containing a scaffolding protein with many attached cellulolytic enzymes. The assembly of such complexes requires engineering of highly specific cohesin-dockerin interactions. Our approach focuses on adapting the surface layer of Caulobacter crescentus for the for highly efficient display of cellulolytic enzymes. We specifically chose C. crescentus as it natively expresses a two dimensional crystal lattice protein called a surface layer (S-Layer), which can be engineered to work as an enzyme display. Our goals were to embed cellulolytic enzymes into the S-Layer protein to confirm their proper folding and activity. We believe that our approach will simplify the process of "cellulosome" engineering by exploiting the robust secretion and display system of Caulobacter. The developed platform can be easily adapted for the display of different enzymatic activities.

Key Acheivements

• Cloned 5 different cellulase constructs, such as β-1,4-endoglucanase (Endo5A), β-1,4-glucosidase (Gluc1C), β-1,4-exoglucanase (CEX) and 2 versions of β-1,4-endoglucanase(E1) in p4A723 plasmid containing rsaA protein and transformed into C. crescentus for the display on cell surface.
• Cloned 4 different cellulases - Endo5A, Gluc1C, E1, G12 into pSB1C3 and submitted as parts BBa……
• Confirmed surface protein (rsaA)-cellulase fusion proteins expression of Endo5A, Gluc1C, E1_422, E1_399 constructs
• Confirmed cellulase activity of Endo5A, Gluc1C, E1_422, E1_399 cellulase constructs expressed on the surface of C. crescentus
• Confirmed baseline intracellular cellulase activity of 4 different cellulase constructs expressed in E. coli: Endo5A, Gluc1C, E1, Gluc1C
• Codon-optimized β-1,4-endoglucanase (cenA) derived from Registry for expression in Caulobacter. Confirmed its expression and cellulase activity on the surface of Caulobacter

Introduction

Plant biomass is the most abundant renewable resource on Earth. Usually plant biomass consists of 30-50% of cellulose, 20-40% hemicellulose carbohydrates and 20-35% lignin heteropolymer, which are integrated in each other to form complex carbohydrate structures recalcitrant to degradation. Cellulose represents a linear polymer of glucose molecules connected through β-1,4 glycoside bonds. In nature the degradation of cellulose into soluble sugars often involves several different enzymes, such as exoglucanases, endoglucanases and β-glucosidases, that act in synergy to cleave the bonds.
We propose to express a mixture of cellulase-degrading enzymes on the surface of the bacteria Caulobacter crescentus,which will work in synergy to increase the rate of degradation and yield of monosaccharides release.

Caulobacter crescentus is a gram negative non-pathogenic bacterium found in many freshwater and soil environments. It displays, on its outer surface, a crystalline protein lattice called a surface layer (S-layer) which is made of multiple repetitions of the same RsaA protein encasing the whole surface of the cell.

For our project we have used the RsaA fusion protein secretion system designed by the Smit lab at the University of British Columbia. Using the ΔGCSS strain of C.crescentus which has its own wild type RsaA protein knock-out in the host genome, we have aimed to insert genetically engineered RsaA fusion protein plasmids to express functional cellulase enzymes fused into the surface layer protein. The use of fusion proteins expressed on the surface of the cell would allow for the breakdown of cellulase without any diffusion of the substrate in and out of the cell.

Design

WE MUST ADD THIS SECTION :)

Methods

All Caulobacter cultures were grown in PYE media supplemented with 2 μg/ml chloramphenicol unless stated otherwise. All plasmid DNA extractions were performed with QIAprep Spin Miniprep Kit (Qiagen). DNA purification from gels or PCR mixtures were done with NucleoSpin® Gel and PCR Clean-up kit(Macherey-Nagel).

Cloning of cellulase enzymes into rsaA plasmid in C. crescentus

The synthesized cellulase DNA was amplified using PCR with HF Phusion polymerase in 100 μl reactions.The following primers were used to amplify the selected region of cellulases genes and to add the BglII and PstI or NheI cut sites for cloning in p4A_723 plasmid:

Endo5A	endo5a_34_fwd	5’-TCCAGATCTAGCGTCAAGGGGTATTACCAC
	endo5a_385_rev	5’-TCCATCTGCAGACACCGGCTTCATGATCCG
Gluc1c	Gluc1c_4_fwd	5’-TCCAGATCTAACACGTTCATCTTTCCGGC
	gluc1c_448_rev	5’-TCCATCTGCAGAGAACCCGTTCTTGGCCAT
E1_399	E1_42_fwd	5’-TCCAGATCTGTTGCAGGCGGGGGTTATTG
	E1_399_rev	5’-TCCATCTGCAGAAACCGGGTCAAATATCGATGATTTTATC
E1_422	E1_42_fwd	5’-TCCAGATCTGTTGCAGGCGGGGGTTATTG
	E1_422_rev	5’-TCCATCTGCAGATGAGGGGGAGGGAGAC
G12	G12_35_fwd	5’-TCCAGATCTGCGACGACCTCCACG
	G12_274_rev	5’-TCCATCTGCAGATGAGGGGGTGGGAGTAG
CEX	JN CEX -1	5’-GGGAGATCTGCGACCACGCTCAAGGAGGCCGCC
	JN CEX - 2	5’-CTAGCTAGCCCCGGCCGGACCGGACGTCGG

For the cloning of Endo5A, GlucIC, G12, E1_422 and E1_399 in P4A723 rsaA plasmid, the amplified products and the isolated p4A723 plasmid DNA were digested with BglII and PstI restriction enzymes (NEB). For the cloning of CEX construct, the plasmid and amplified product were digested with BglII and NheI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase(NEB) and the ligation mixes were then transformed in chemically competent DH5α E. coli.

Transformed colonies were then plated on and selected from a LB-CM plate and grown overnight in LB-CM media. Recombinant plasmid was then isolated and sent for Sanger sequencing for confirmation.

After sequence confirmation, the isolated construct DNA were electroporated into electrocompetent C.crescentus and colonies were grown on a PYE-CM plate. One colony was selected and streaked onto a fresh plate, which would be used for all future assays.

Cloning of cellulase enzymes into pSB1C3 plasmid with Ptac promoter and rbs

The synthesized cellulases genes were amplified using PCR with HF Phusion polymerase in 100 μl reactions.The following primers were used to amplify the selected region of cellulases genes and to add BioBrick Prefix and Suffix: Endo5A Primers 5'-TCCgaattcgcggccgcttctagATGAAGAAGAAGGGC and 5'-TCCtactagtagcggccgctgcagCTACTCCGCGGAGG, Gluc1C Primers 5'-TCCgaattcgcggccgcttctagATGAGCGAGAACACG and 5'-TCCtactagtagcggccgctgcagTTAGAACCCGTTCTTG, E1 Primers 5'-TCCgaattcgcggccgcttctagATGCCTCGCGCTCT and 5'-TCCtactagtagcggccgctgcagCTAGGTGGGAGTTGGG, G12 Primers 5'-TCCgaattcgcggccgcttctagATGTTAGTGTTAAGAGCC and 5'-TCCtactagtagcggccgctgcagCTACGACGAAGTAGG

Purified pSB1C3 plasmid and the amplified Endo5A, Gluc1C, E1, G12 constructs were digested with EcoRI and SpeI restriction enzymes. The plasmid digests were purified by agarose gel purification, while PCR product digests were purified by PCR purification. Purified digests were ligated using T4 ligase and the ligation mixes were then transformed in chemically competent DH5α E. coli. Transformed colonies were then plated on LB-CM plate and the isolated colonies were selected and grown overnight in LB-CM media. Recombinant plasmid was isolated and sent for Sanger sequencing for confirmation.

pTAC was amplified from Bba_K868400 part using HF phusion PCR with the following primers to add the rbs (part Bba_B0034): Ptac_rbs_F 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAgagctgttgacaatta, Ptac_rbs_R 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGT tttctcctcttt aattgttatccgctca

The amplified pTAC and rbs part was then digested with EcoRI and SpeI restriction enzymes. pSB1C3 high copy assembly plasmid with the gene insert was digested using EcoRI and XbaI restriction enzymes. The plasmid digest was then purified by agarose gel purification while the pTAC RBS digest was purified by PCR purificationt. Both purified digest were ligated together using standard ligation protocol and ligation mix was then transformed in chemically competent dh5a E.coli.

Surface layer fusion protein extraction

10 mL tubes of PYE-CM were inoculated with the different C. crescentus strains carrying the different cellulase-rsaA fusions in p4A723 plasmid as well as a positive control carrying a plasmid with wild type rsaA (P4A723) and a negative control of rsaA knock-out (ΔGCSS). Cultures were grown overnight at 30°C shaking. Cultures were taken out of incubator and optical density at 600 nm was measured. All cultures were then normalized to the lowest OD by diluting the remaining culture with PYE.
Low pH extraction (Walker et al. 1992) was performed using different pHs of HEPES buffer to extract only crystalline s-layer without lysing the cells. Proteins were then stored in eppendorf tubes at -20°C. Remaining cell pellet was used to prepared a cell lysate by resuspending the pellet in 150 ul of lysis buffer and boiling for 5 minutes.

Surface layer fusion protein expression confirmation

To confirm expression of surface layer fusion protein SDS-PAGE and western blot analysis were performed on proteins from low pH extraction and on the cell lysates from those samples. SDS-PAGE was done using 8% separating gels. Coomassie Brilliant Blue staining and western immunoblotting was performed using protocols outlined in protocol section. Western blots were probed with primary rabbit anti-RsaA polyclonal antibodies at 1/30,000 dilution. Goat anti-rabbit IgG was used as secondary antibody at 1/50,000 dilution. Fluorophore was detected by Odyssey Infrared Imaging System.

Caulobacter Cellulase Activity Analysis

For cellulase enzyme activity measurement, triplicate 5mL PYE-CM starter cultures were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD 600 nm by diluting the remaining culture with PYE. 1 mL of each sample was removed and resuspended with fresh PYE and spin down 3 times to remove any lysed cells that may interfere with cellulase activity results.

A protocol for cellulases activity assay was adopted from “A high throughput screen for biomining cellulase activity from metagenomic libraries”. 150 uL of each culture were aliquoted into a clear 96 well plate(Corning) then 150 uL assay mix (0.1 mg/ml DNPC in 0.1 M pH 5.5 potassium acetate buffer) was added to the each well. OD 400 nm and OD 600 nm was measured every 30 minutes for several hours by a plate reader. Between measurements the culture was incubating at 30°C. Data was normalized further by dividing the OD400nm measurement by the OD600nm measurement.

OD 400 nm was measured every 30 minutes for 5 hours by an XX plate reader. Between measurements the culture was incubating at 30°C.

Growth of Caulobacter displaying cellulases on cellulose as a sole carbon source

Triplicate 5mL PYE-CM starter cultures of p4A723, E1_399, E1_422, Gluc1C and Endo5A were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining culture with PYE. 100-fold dilution was used to inoculate the cultures in 200 uL well clear plate containing M2 with 0.2% w/v carboxymethylcellulose. For the co-cultures, equal-volume inoculations were performed to make final dilution of 100. The plate was incubate at 30C in a Tecan platereader and OD600 was read every 30 minutes.

Results

Cloning of cellulase enzymes into rsaA plasmid in C.crescentus

Endo5A, Gluc1C, E1_422, and E1_399 were all successfully cloned into the p4A723 plasmid vector and sequence confirmed by Sanger sequencing. Cloning was attempted on G12 several times, each time the transformation step into DH5α E.coli failed, leading to the assumption that the gene product might be toxic to the cell. As Endo5A, E1_422, and E1_399 are all endoglucanases, we decided to not proceed with any troubleshooting on G12. CEX gene was successfully cloned into DH5α E.coli and sequence confirmed. However after transforming the plasmid into caulobacter, extreme cell clumping was observed, possibly a result of the CEX fusion into the S-Layer.

Surface layer fusion protein extraction expression confirmation

Low pH extraction was done on Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusion proteins as well as P4A732 RsaA positive control and ΔGCSS negative control. We also prepared a cell lysate from the remaining cell pellet to compare the amount of protein within the cell as opposed to on the surface of the cell. We ran two gels simultaneously staining one with Coomassie Brilliant blue and performing western blot on the other.
Thermo fisher prestained protein ladder was used to compare relative expected sizes of fusion proteins. Expected protein sizes were as followed:

Protein	Size
RsaA	98 kDaltons
Endo5A-RsaA fusion	138 kDaltons
Gluc1C-RsaA fusion	149 kDaltons
E1_399-RsaA fusion	138 kDaltons
E1_422-RsaA fusion	140 kDaltons

On the coomassie stained gel faint bands corresponding to cellulases-rsaA fusionswere observed in low PH extracted proteins. The cell lysate showed large amounts of intracellular proteins with poor differentiation so no conclusions could be made from these.
On the western blot analysis, RsaA fusion proteins were clearly observed at higher molecular weight than wild type RsaA. Nothing was displayed on the negative control showing that no wild type RsaA was produced. The cell lysate results were comparable to the low pH extracted proteins, with visibly more RsaA fusion proteins detectable in the cell lysate as opposed to the low pH extracted proteins. This may be a sign that not all of the fusion protein can get out of the cell and crystalize, likely due to the large size of the fusion protein. The higher visibility of some constructs over others in the low pH extraction, such as with E1_399 vs. Gluc1C, is indicative of higher expression and secretion outside of the cell. The smaller constructs such as E1_399 and Endo5A were seen more clearly on both the coomassie stained gel and the western blot, indicating that the smaller constructs might be easier to transport on the surface. Some fragments of lower molecular weight RsaA protein in the western, this is probably due to cleavage of the RsaA protein during the extraction.

Caulobacter cellulase activity analysis

An initial cellulase activity analysis was run using the above protocols for a period of 150 minutes. The assay mechanisms is explained in Fig. X. 2,4dinitrophenylcellobiose consists of cellobiose bound to chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400

This assay was run on the Caulobacter cells expressing Endo5A, Gluc1C, E1_399, and E1_422 RsaA fusions using the wild type RsaA P4A723 protein as a control. From the first time point, cellulase-expressing Caulobacters demonstrated high cellulase activities, with E1_422 and E1_399 having the highest activity level followed by Endo5A (Figure XX). Gluc1C had lower activity levels closer to that of the control, what can be explained by the glucosidase activity of Gluc1C. The enzyme cleaves a glucose molecule from DNPC first, not cellobiose what does not lead to the release of chromophore. Observation at later time points need to be taken to confirm the Gluc1C activity.

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Conclusion

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References

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