LCC: A variety of cloning efforts
Leaf-branch compost cutinase (LCC) is a cutinase homolog enzyme first discovered and isolated by Sulaiman et al. in 2012 through a metagenomic study from leaf-branch compost.
Compost is known to contain many plant-cell degrading microorganisms, presenting a potential source of novel and promising genes encoding enzymes with cutinase activities.
During active composting the temperature of leaf-branch compost reaches up to 70 °C and therefore the enzymes functioning in the compost are very thermostable, making them desirable for many industrial applications.
After its discovery LCC was recombinantly expressed in E. coli with PelB signal sequence, which resulted in extracellular secretion of the enzyme and secreted LCC showed PET-degrading activity.
The specific PET-degrading activity of LCC was the highest of all known cutinases documented so far and instantly captured the attention of many researchers.
LCC is a monomeric enzyme and besides PET hydrolyzes various monoesters of fatty acid, most efficiently at pH 8.5 and 50 °C (Sulaiman et al. 2014).
Studies showed that LCC was at least 33 times more active than all other enzymes, previously reported to hydrolyse PET, with its PET-specific depolymerization rate of 93.2 mgTAeq h-1 mgenzyme-1 at 65 °C, while also demonstrating the highest thermostability (Tournier et al.. 2020).
Binding of PET in the active site protects the site from conformational changes due to high temperature and therefore stabilises the site and increases activity at higher temperatures (Sulaiman et al.. 2014).
The resolved LCC crystal structure showed a catalytic triad formed by residues Ser165, Asp210, and His242 and a disulfide bond formed between Cys280 and Cys298, which increases stability and kinetic robustness of the enzyme.
LCC denatures at around 86 °C and the optimal temperature for PET degradation is around 70 °C, the temperature of PET glass transition.
For PET degrading enzymes it is important to function above the PET glass transition temperature, where PET chains are the most flexible.
Several attempts were made to increase LCC thermal stability and kinetic activity.
Shirke et al.. expressed the enzyme in Pichia pastoris, where it was N-glycosylated and resulted in increased stabilisation against thermal aggregation and in improved catalytic PET hydrolysis.
LCC was also stabilised through addition of a new disulfide bond (D238C/S283C), increasing the melting temperature by almost 10 °C, and mutated to produce a more catalytically active enzyme.
We chose to try to produce LCC enzyme in our project due to its efficiency as a promising alternative to PET degradation by PETase.
LCC could be used at high temperatures as a pretreatment of PET.
Three attempts to create a functioning LCC gene were made with the following DNA constructs.
Attempt 1
Coding sequence for LCC enzyme was taken from GenBank: AEV21261.1 with codon-optimised nucleotide sequence for expression in E. coli. BioBrick prefix, containing restriction sites for EcoRI and XbaI, and suffix with sites for restriction by SpeI and PstI were added on ends of the construct for easier insertion into vectors. Our construct was also designed with an RBS, T7 promoter and terminator, lac operator for optimal induction of protein expression (Shepherd et al. 2017, Du et al. 2012) and a C-terminal histidine tag for enzyme purification on immobilized metal affinity chromatography (Figure 1). Construct was created in Snapgene and then ordered. The construct did not include buffer nucleotides on the DNA ends and therefore they could not be digested with EcoRI and PstI restriction enzymes and directly inserted into a BioBricked pET24a plasmid (Du et al. 2009). Primers for BioBrick sites were ordered to create overhangs in the LCC construct. PCR primers used were: Forward 5’-NNNNNGGAATTCGCGGCCGCTTCTAG, Reverse 5’-NNNNNNCTGCAGCGGCCGCTACTA-3’.
Figure 1: Snapgene schema of the ordered LCC insert for attempt 1. BioBrick prefix, T7 promoter, lac operator and RBS precede the coding sequence and C-terminal histidine tag, T7 terminator and BioBrick suffix follow after the coding sequence.
Attempt 2
Our second attempt for LCC recombinant expression relied on parts from the 2019 iGEM distribution kit. Two constructs were obtained from the kit: LCC (BBa_K936000) and LCC pelB (BBa_K936013) on psb1C3 plasmid. PelB tag is a signal peptide used for better secretion of the expressed enzyme outside of the cell (Shi et al. 2021). To successfully express the LCC enzyme RBS+T7 promoter and T7 terminator needed to be inserted into the plasmids with the LCC or LCC pelB gene. Both parts were obtained from the 2019 iGEM distribution kit (RBS+T7 promoter on psb1C3: BBa_K525998 and T7 terminator on psb1C3: BBa_K731721). T7 terminator was afterwards also used from 2017 iGEM distribution kit and on a plasmid psb1C3TΦ obtained from our teaching assistant Letian Bao, who also provided plasmid psb1K3J04450 with an inserted gene for red fluorescent protein (mRFP) (Bao et al. 2020, Liljeruhm et al. 2018 ).
Attempt 3
For our final attempt to produce a functioning plasmid with the LCC gene, we ordered a new insert. In addition to the same sequence as the first ordered insert it also contained buffer nucleotides on each end to provide enough space for restriction enzymes to work properly (Figure 2).
Figure 2: Snapgene schema of the ordered LCC insert for attempt 3. BioBrick prefix, T7 promoter, lac operator and RBS precede the coding sequence and C-terminal histidine tag, T7 terminator and BioBrick suffix follow after the coding sequence. Parts highlighted in red are buffer nucleotides differing the insert from the one used in attempt 1.
Attempt 1
Three different PCR methods (touchdown, gradient and limited gradient) were tested to produce BioBricked LCC construct with overhangs of buffer nucleotides.
PCR elongations were set for 40 s due to LCC construct length around 1000 bp.
Agarose gel analysis with SybrSafe staining showed that none of the methods provided any PCR product (Figure 3-4).
Figure 3: Agarose gel analysis of touchdown PCR, with LCC product expected at the size of 1 kb, which does not show any product. Samples in respective wells: L-ladder, PT-PETase PCR touchdown analysis, LT- LCC PCR touchdown analysis.
Figure 4: Agarose gel analysis of gradient PCR (upper gel) and limited gradient PCR (lower gel) with LCC product expected at the size of 1 kb, which does not show any product in any analysed samples.
Repeated gradient PCR resulted in some amplification in the expected LCC region. The respective gel band was therefore purified (GeneJET Gel Extraction Kit from Thermo Scientific), and obtained DNA was digested with EcoRI and PstI endonucleases and ligated into a BioBricked pET24a and psC1B3 vectors. Attempts to transform these vectors into competent cells were however unsuccessful and could not be repeated due to insufficient amount of produced LCC insert.
Attempt 2
Constructing a functional plasmid from separate iGEM parts was tried in our second attempt. Both LCC and LCC pelB constructs were successfully transformed into competent DH5α cells. After amplification in cells, plasmids were extracted with GeneJET miniprep kit from Thermo Scientific.
To successfully express the LCC enzyme, RBS+ T7 promoter and T7 terminator needed to be inserted into the plasmids with LCC (or LCC pelB) gene.
RBS+T7 promoter construct was amplified in DH5α cells and extracted. T7 terminator plasmid produced no colonies after transformation into DH5α cells.
A new attempt was then made with the T7 terminator part from the 2017 iGEM distribution kit, which also produced no colonies.
Plasmid psb1C3TΦ was finally used in the following experiments.
In the first attempt to ligate LCC (or LCC pelB) with RBS+ T7 promoter, LCC (or LCC pelB) plasmid were digested with XbaI and PstI; and plasmid with RBS+ T7 promoter was restricted with SpeI and PstI (all restriction enzymes were from NEB).
Restricted segments were separated on an agarose gel (10 % agarose with SybrSafe staining), desired DNA was extracted from the gel (GeneJET Gel Extraction Kit from Thermo Scientific) and ligated with T4 DNA ligase from NEB.
However, restriction sites for SpeI and PstI are too close together to be successfully restricted on both sites and therefore this approach did not work.
A 3A assembly then had to be performed to insert LCC (or LCC pelB) into a plasmid together with the RBS+T7 promoter.
Backbone plasmid psb1K3J04450 with inserted gene for red fluorescent protein (mRFP) was then digested with EcoRI and PstI, LCC (or LCC pelB) plasmid was restricted with XbaI and PstI and RBS+ T7 promoter plasmid was cut with EcoRI and SpeI.
Restricted segments were ligated and transformed into DH5α cells, which produced some colonies expressing mRFP, where the ligation was unsuccessful, but also a few colourless colonies, with our product (Figure 5).
Figure 5: Kanamycin plate with transformed DH5α cells containing product of first 3A assembly. Marked colonies contained religated backbone plasmid psb1K3 with gene for fluorescent mRFP protein and unmarked colonies contained no fluorescent protein and were therefore considered as successful ligation products containing LCC pelB gene with RBS and T7 promoter on a backbone of psb1K3 plasmid.
Plasmids from two successful colonies were amplified and extracted and 3A assembly was repeated with our new plasmids containing LCC (or LCC pelB) and RBS+ T7 promoter (cut with EcoRI and SpeI), psb1K3J04450 as backbone (cut with EcoRI and PstI) and psb1C3TΦ containing T7 terminator (cut with XbaI and PstI).
Ligated products were transformed into DH5α cells and produced several red colonies, where backbone plasmid religated with the mRFP gene, and some colourless colonies with ligated product.
Restriction digestion with EcoRI and PstI was done to analyse the final insert in the plasmid and results were visualised on a 10 % agarose gel.
The analysis showed that the final plasmid has around 2 kbp compared to expected 1 kbp (calculated size of LCC + RBS + T7 promoter + T7 terminator) and therefore our final product does not contain the desired LCC insert (Figure 6).
Figure 6: Agarose gel analysis of digested plasmids after second 3A assembly. The desired product was expected at 1 kb size after restriction with EcoRI and PstI. None of the selected colonies contained a plasmid, which would be at the expected size after restriction. None of the colonies therefore contain the expected product and no further experiments were carried out with these constructs. Samples in respective wells: L-ladder, LCC col I- plasmid from colony one, which should contain full plasmid with LCC gene without restriction, LCC col I E+P- plasmid from colony one, which should contain full plasmid with LCC gene digested with EcoRI and PstI, LCC col II- plasmid from colony two, which should contain full plasmid with LCC gene without restriction, LCC col II E+P- plasmid from colony two, which should contain full plasmid with LCC gene digested with EcoRI and PstI, LCC pelB col I- plasmid from colony one, which should contain full plasmid with LCC gene without restriction, LCC pelB col I E+P- plasmid from colony one, which should contain full plasmid with LCC gene digested with EcoRI and PstI, LCC pelB col II- plasmid from colony two, which should contain full plasmid with LCC gene without restriction, LCC pelB col II E+P- plasmid from colony two, which should contain full plasmid with LCC gene digested with EcoRI and PstI.
Attempt 3
Our new insert with buffer nucleotides as well as pET24a vector were restricted with EcoRI and PstI and ligated together. Ligated product was transformed into DH5α cells and produced 4 colonies in total, which were all analysed using colony PCR (extension time: 16 s) followed by agarose gel separation and none of the colonies contained plasmid with inserted LCC (Figure 7).
Figure 7: Agarose gel of a colony PCR of four colonies picked from a plate after transformation of LCC gene into a pET24a vector. Expected size of the amplified product would be around the size of 1 kb and none of the colonies provided such a product. Sample in well 1 probably contained amplified IF3 gene around the size of 0.8 kb. Samples in respective wells: L-ladder, Col I- amplified plasmid insert from colony 1, Col II- amplified plasmid insert from colony 2, Col III- amplified plasmid insert from colony 3, Col IV- amplified plasmid insert from colony 4, pET24a IF3- amplified plasmid insert from colony transformed with pET24a vector containing BioBricked IF3 gene insert, H2O- negative control for PCR reaction, where water was added instead of a bacterial colony.
Bao L, Menon PNK, Liljeruhm J, Forster AC. 2020. Overcoming chromoprotein limitations by engineering a red fluorescent protein. Analytical Biochemistry 611: 113936.
Du L, Gao R, Forster AC. 2009. Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase. Biotechnology and Bioengineering 104: 1189–1196.
Du L, Villarreal S, Forster AC. 2012. Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase. Biotechnology and Bioengineering 109: 1043–1050.
Liljeruhm J, Funk SK, Tietscher S, Edlund AD, Jamal S, Wistrand-Yuen P, Dyrhage K, Gynnå A, Ivermark K, Lövgren J, Törnblom V, Virtanen A, Lundin ER, Wistrand-Yuen E, Forster AC. 2018. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 12: 8.
Shepherd TR, Du L, Liljeruhm J, Samudyata null, Wang J, Sjödin MOD, Wetterhall M, Yomo T, Forster AC. 2017. De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research 45: 10895–10905.
Shi L, Liu H, Gao S, Weng Y, Zhu L. 2021. Enhanced Extracellular Production of IsPETase in Escherichia coli via Engineering of the pelB Signal Peptide. Journal of Agricultural and Food Chemistry 69: 2245–2252.
Shirke AN, White C, Englaender JA, Zwarycz A, Butterfoss GL, Linhardt RJ, Gross RA. 2018. Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis. Biochemistry 57: 1190–1200.
Sulaiman S, Yamato S, Kanaya E, Kim J-J, Koga Y, Takano K, Kanaya S. 2012. Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach. Applied and Environmental Microbiology 78: 1556–1562.
Sulaiman S, You D-J, Kanaya E, Koga Y, Kanaya S. 2014. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase. Biochemistry 53: 1858–1869.
Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux M-L, Texier H, Gavalda S, Cot M, Guémard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, André I, Duquesne S, Marty A. 2020. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580: 216–219.
