Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product

Background: Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. Results: The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. Conclusions: The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg Inr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization