Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Blanusa, Milan; Schenk, Alexander; Sadeghi, Hengameh; Marienhagen, Jan; Schwaneberg, Ulrich

doi:10.1016/j.ab.2010.07.011

Cited by 107 publications

(110 citation statements)

References 16 publications

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“…The decreased translation efficiency of the other enzymes possibly decreases the metabolic burden for the microbial host with respect to protein synthesis, thus balancing the whole heterologous pathway. The absolute product titer of the best five variants ranged from 47.6 to 52 mg/L All variants of the operon were cloned into the pALXtreme vector backbone [16] (general scheme: pALXtreme-1a_T7 prom >x-Rstal-x-Pc4cl-x-Zmccr-x-Zmcad) and subsequently transformed to E. coli BL21 (DE3) lacI Q1 [relevant characteristics of the genotype: 11 5-9-5-9 38 9-9-5-9 65 13-9-5-9 12 5-9-5-13 39 9-9-5-13 66 13-9-5-13 13 5-9-9-5 40 9-9-9-5 67 13-9-9-5 14 5-9-9-9 41 9-9-9-9 68 13-9-9-9 15 5-9-9-13 42 9-9-9-13 69 13-9-9-13 16 5-9-13-5 43 9-9-13-5 70 13-9-13-5 17 5-9-13-9 44 9-9-13-9 71 13-9-13-9 18 5-9-13-13 45 9-9-13-13 72 13-9-13-13 23 5-13-9-9 50 9-13-9-9 77 13-13-9-9 24 5-13-9-13 51 9-13-9-13 78 13-13-9-13 25 5-13-13-5 52 9-13-13-5 79 13-13-13-5 26 5-13-13-9 53 9-13-13-9 80 13-13-13-9 27 5-13-13-13 54 9-13-13-13 81 13-13-13-13…”

Section: Resultsmentioning

confidence: 99%

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli

et al. 2016

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Background: Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Escherichia coli.Results: Basis for the construction of a p-coumaryl alcohol producing strain was the development of Operon-PLICing as method for the rapid combinatorial assembly of synthetic operons. This method is based on the chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution to generate complementary, single-stranded overhangs and subsequent hybridization of multiple DNA-fragments. Furthermore, during the assembly of these DNAfragments, Operon-PLICing offers the opportunity for balancing gene expression of all pathway genes on the level of translation for maximizing product titers by varying the spacing between the Shine-Dalgarno sequence and START codon. With Operon-PLICing, 81 different clones, each one carrying a different p-coumaryl alcohol operon, were individually constructed and screened for p-coumaryl alcohol formation within a few days. The absolute product titer of the best five variants ranged from 48 to 52 mg/L p-coumaryl alcohol without any further optimization of growth and production conditions. Conclusions:Operon-PLICing is sequence-independent and thus does not require any specific recognition or target sequences for enzymatic activities since all hybridization sites can be arbitrarily selected. In fact, after PCR-amplification, no endonucleases or ligases, frequently used in other methods, are needed. The modularity, simplicity and robustness of Operon-PLICing would be perfectly suited for an automation of cloning in the microtiter plate format.

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Section: Resultsmentioning

confidence: 99%

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli

et al. 2016

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“…CLC Main Workbench software (Qiagen) was employed for sequence assembly, BLASTX analysis, and identification of ORFs. The esterase-coding genes for EstB11, EstC5, EstC7, EstC9, and EstG4 were cloned into pET28a(ϩ) (Novagen, USA) through restriction cloning, while EstB3 was cloned with a ligase-independent method (phosphorothioate-based ligaseindependent gene cloning [PLICing]), as described elsewhere (51). A standard PCR mixture (50 l) contained 1ϫ Phusion HF buffer, 1 U of Phusion DNA polymerase (New England BioLabs), 0.2 mM deoxynucleoside triphosphates, 0.2 M each primer, and 10 ng of plasmid DNA.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Polyesterases from Moss-Associated Microorganisms

Müller

Perz

Provasnek

et al. 2017

Appl Environ Microbiol

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The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli. The esterases were found to hydrolyze the copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p-nitrophenyl butyrate (K m values of 46.5 and 3.4 M, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds.IMPORTANCE In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material.

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“…Step 2 involves chemical cleavage of phosphorothiolated nucleotides to generate complementary DNA overhangs on the 5′-ends of each DNA fragment [44,45].…”

Section: Tmgs-pcr [95]mentioning

confidence: 99%

“…• TrimerDimer [38] • SILM [40] • OmniChange [9] • SySM [44] • Amber Codon [49] • MCST [55] • dITP-epPCR [60] • POE-PCR [61] • MegAnneal [58] • TRINS [54] • epRCA [56] • SeSaM-III [59] • TMGS-PCR [92] • USERec [96] • GoldenGate Shuffling [95] • PTRec [91] • Integron [94] Covered only 48 clones revealed 66-84% coverage of all possible codons at all five targeted positions (up to 27 out of 32 possible codons). OmniChange fulfills all the requirements in terms of robustness and simplicity with respect to becoming a successful method for focused multisite saturation mutagenesis.…”

Section: Focused Mutagensis II Random Mutagensis Iii Dna Recombimentioning

confidence: 99%

To get what we aim for – progress in diversity generation methods

2013

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Protein re‐engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high‐throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized ‘to get what we aim for’ through ‘optimal diversity’ generation.

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Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Cited by 107 publications

References 16 publications

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli

Discovery of Polyesterases from Moss-Associated Microorganisms

To get what we aim for – progress in diversity generation methods

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