title: Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.
abstract: Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.
authors.affiliation: Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Rivera-Hoyos
authors.affiliation: Laboratorio de Microbiología Ambiental y Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia; Departamento de Química y Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales-Caldas, Colombia.
authors.forename: Edwin David
authors.initials: ED
authors.lastname: Morales-Álvarez
authors.affiliation: Escuela de Ciencias Biológicas, Facultad de Ciencias Básicas, Universidad Pedagógica y Tecnológica de Colombia (UPTC), Tunja-Boyacá, Colombia.
authors.forename: Sergio Alejandro
authors.initials: SA
authors.lastname: Poveda-Cuevas
authors.affiliation: Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia (UNAL), Bogotá, D.C., Colombia.
authors.forename: Edwin Alfredo
authors.initials: EA
authors.lastname: Reyes-Guzmán
authors.affiliation: Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
authors.forename: Raúl A
authors.initials: RA
authors.lastname: Poutou-Piñales
authors.affiliation: Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia (UNAL), Bogotá, D.C., Colombia.
authors.forename: Edgar Antonio
authors.initials: EA
authors.lastname: Reyes-Montaño
authors.affiliation: Laboratorio de Microbiología Ambiental y Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
authors.forename: Aura Marina
authors.initials: AM
authors.lastname: Pedroza-Rodríguez
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigaciones y de Estudios Avanzados del Instituto Politécnico Nacional (IPN), México, D.F., México.
authors.forename: Refugio
authors.initials: R
authors.lastname: Rodríguez-Vázquez
authors.affiliation: Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
authors.forename: Ángela M
authors.initials: ÁM
authors.lastname: Cardozo-Bernal
mesh_terms: D003198:Computer Simulation; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D042845:Laccase / Q000096:biosynthesis / Q000235:genetics; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D020076:Pleurotus / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020940:Reishi / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D042845:Laccase