Recombinant Production Processes Research Articles

Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria

BackgroundLactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications.ResultsTo evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates.ConclusionsThese results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.

A novel membrane stirrer system enables foam-free biosurfactant production.

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble-free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane-module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam-free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3-L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTRmax ) of 175 mmol L-1 h-1 in completely foam-free cultivations. With a high space-time yield (STY) of 118 mgRL L-1 h-1 during a fed-batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.

Recombinant production of the lantibiotic nisin using Corynebacterium glutamicum in a two-step process

BackgroundThe bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products.ResultsWe established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin.ConclusionsWe demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

Recombinant human C1 esterase inhibitor (Conestat alfa) for prophylaxis to prevent attacks in adult and adolescent patients with hereditary angioedema

ABSTRACTIntroduction: Hereditary angioedema (HAE) due to C1 inhibitor (C1-INH) deficiency is a debilitating and potentially lethal disease. Management includes on-demand treatment of angioedema and their prophylaxis. Plasma derived C1-INH is an established treatment for both on demand and prophylaxis of HAE. Conestat alfa is a recombinant form of human C1-INH (rhC1-INH) produced in transgenic rabbits. It has granted drug’s registration as treatment option for acute HAE attacks in adults and adolescents in Europe, America, and other countries. Long-term prophylaxis with rhC1-INH received recent consideration in clinical trials.Areas covered: This review will critically appraise available information about rhC1-INH (conestat alfa) prophylactic treatment in adult and adolescent patients with congenital C1-INH deficiency. Results from a phase II randomized placebo-controlled trial for prophylaxis of severe HAE evidenced positive treatment outcomes for its application, both twice or once weekly.Expert commentary: Phase II clinical studies suggest that rhC1-INH is a viable option for prophylaxis of HAE. Safety and tolerability data are comparable to other available HAE specific drugs, zeroing the possibility for blood-born viral transmission. Sustainability of modern technologies is granting a practically stable and continuous recombinant production process. With other available options, rhC1-INH facilitates tailoring HAE treatment to patients’ needs.

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics

BackgroundPreclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1–1 versus Hp 2–2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200–900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions.ResultsIn the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies.ConclusionsWe present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.

Ultrastrong and Bioactive Nanostructured Bio-Based Composites.

Nature's design of functional materials relies on smart combinations of simple components to achieve desired properties. Silk and cellulose are two clever examples from nature-spider silk being tough due to high extensibility, whereas cellulose possesses unparalleled strength and stiffness among natural materials. Unfortunately, silk proteins cannot be obtained in large quantities from spiders, and recombinant production processes are so far rather expensive. We have therefore combined small amounts of functionalized recombinant spider silk proteins with the most abundant structural component on Earth (cellulose nanofibrils (CNFs)) to fabricate isotropic as well as anisotropic hierarchical structures. Our approach for the fabrication of bio-based anisotropic fibers results in previously unreached but highly desirable mechanical performance with a stiffness of ∼55 GPa, strength at break of ∼1015 MPa, and toughness of ∼55 MJ m-3. We also show that addition of small amounts of silk fusion proteins to CNF results in materials with advanced biofunctionalities, which cannot be anticipated for the wood-based CNF alone. These findings suggest that bio-based materials provide abundant opportunities to design composites with high strength and functionalities and bring down our dependence on fossil-based resources.

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme.

Clonación, expresión y caracterización de una nueva esterasa derivada de metagenomas de suelos agrícolas colombianos

El presente trabajo tuvo como objetivo la bioprospección de ADN metagenómico derivado de comunidades microbianas asociadas a un agroecosistema de importancia nacional. Este análisis permitió realizar la producción, expresión, purificación y caracterización de una enzima novedosa con actividad esterasa. Esta enzima, denominada LipM, había sido previamente identificada en clones metagenómicos derivados de suelos dedicados al cultivo de papa criolla (Solanum pureja), mediante secuencia de nueva generación y análisis bioinformáticos. La secuencia codificante de la enzima fue clonada en el vector pBADgiii y expresada en E. coli como sistema de expresión, lo que permitió optimizar el proceso de producción recombinante y su posterior purificación.Funcionalmente la enzima presentó una mayor afinidad por sustratos de p-nitrofenil con ácidos grasos de cadena corta (<C8). LipM mostró completa funcionalidad a temperaturas entre 30 – 37 ºC y en valores de pH cercanos al fisiológico (entre 7.0 y 8.0). Igualmente, esta enzima exhibió buena estabilidad en presencia de varios iones metálicos, inhibidores y 0.1% (p/v) de SDS. Su alto nivel de estabilidad en presencia de iones metálicos e inhibidores, así como su particular especificidad en cuanto a sustratos, la hacen una enzima óptima para utilización en diferentes aplicaciones biotecnológicas.Palabras clave: metagenómica, enzima esterasa, caracterización, suelos, Lipasa/esterasa

Possibilities of Using Fetal Hemoglobin as a Platform for Producing Hemoglobin-Based Oxygen Carriers (HBOCs).

The expression levels of fetal hemoglobin (HbF) in bacterial recombinant systems are higher compared with normal adult hemoglobin (HbA). However, heme disorientation in globins are often observed in recombinant production processes, both for HbA and HbF, although the degree of heme oriental disorder is much lower for HbF. In addition, the heme disorientation can be converted to a normal conformation by an oxidation-reduction process. A chromatographic cleaning process involving a strong anion exchanger can be utilized to remove such unstable and nondesirable forms of Hb.

Recombinantly produced hydrophobins from fungal analogues as highly surface-active performance proteins

Hydrophobins are available from natural resources only in milligram amounts. BASF succeeded in a recombinant production process, up-scaled to pilot plant production in kilogram scale. Strain and protein optimization by modulation of gene expression and generation of fusion proteins finally leads to two class I hydrophobins called H*Protein A and H*Protein B. By analytical ultracentrifugation, we confirm that the self-association of H*Proteins in solution is governed by their sequence, because oligomerization is induced by the same mechanisms (pH > 6, temperature >> 5 degrees C, concentration > 0.2 mg/ml) as for the well-known native hydrophobins SC3 and HFB II. Additionally, we established the triggering of structure formation by bridging with divalent ions and the stabilization of dimers and tetramers by monovalent ions or surfactants. This interplay with surfactants can be exploited synergistically: The capacity for emulsification of a 300 ppm standard surfactant solution is boosted from 0 to 100% by the addition of a mere 1 ppm of our new hydrophobins, with H*Protein A and H*Protein B having specific application profiles. This astonishing performance is rationalized by the finding that the same minute admixtures enhance significantly the interfacial elastic modulus, thus stabilizing interfaces against coalescence and phase separation.

The application of recombinant human collagen in tissue engineering.

Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cerevisiae.

This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cereriviae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.