title: A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production.
abstract: The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.
authors.affiliation: Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Munjal
authors.affiliation: Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Kamran
authors.initials: K
authors.lastname: Jawed
authors.affiliation: Centre for Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi, India.
authors.forename: Saima
authors.initials: S
authors.lastname: Wajid
authors.affiliation: Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India; DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Syed Shams
authors.initials: SS
authors.lastname: Yazdani
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D056804:Biofuels / Q000382:microbiology*; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D006868:Hydrolysis; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D056804:Biofuels; D004274:DNA, Recombinant; D002475:Cellobiose; D001617:beta-Glucosidase; D002480:Cellulase