Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification.

Background: The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express.

Coenzyme Q Biosynthesis Established in the Non-Ubiquinone Containing by Metabolic Engineering.

Coenzyme Q (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown.

Recombinant expression of an l-amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation.

l-amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His-tag and compare this with the recently characterized 6His-hcLAAO4 expressed in E. coli.

Fermentative -Methylanthranilate Production by Engineered .

The -functionalized amino acid -methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid -isopropyl -methylanthranilate, the flavor compound -methyl--methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to -alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium are operated industrially at the million tons per annum scale.

Bromination of L-tryptophan in a Fermentative Process With .

Brominated compounds such as 7-bromo-l-tryptophan (7-Br-Trp) occur in Nature. Many synthetic and natural brominated compounds have applications in the agriculture, food, and pharmaceutical industries, for example, the 20S-proteasome inhibitor TMC-95A that may be derived from 7-Br-Trp. Mild halogenation by cross-linked enzyme aggregates containing FAD-dependent halogenase, NADH-dependent flavin reductase, and alcohol dehydrogenase as well as by fermentation with recombinant expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from have recently been developed as green alternatives to more hazardous chemical routes.

Fermentative Production of -Alkylated Glycine Derivatives by Recombinant Using a Mutant of Imine Reductase DpkA From .

Sarcosine, an -methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. -methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. , which is used for the industrial production of amino acids for decades, has recently been engineered for production of -methyl-L-alanine and sarcosine.

Fermentative Production of -Methylglutamate From Glycerol by Recombinant .

-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. -alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics.

Efficient Production of the Dicarboxylic Acid Glutarate by via a Novel Synthetic Pathway.

The dicarboxylic acid glutarate is an important building-block gaining interest in the chemical and pharmaceutical industry. Here, a synthetic pathway for fermentative production of glutarate by the actinobacterium has been developed. The pathway does not require molecular oxygen and operates via lysine decarboyxylase followed by two transamination and two NAD-dependent oxidation reactions.

One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst.

N-methylated amino acids are found in Nature in various biological compounds. N-methylation of amino acids has been shown to improve pharmacokinetic properties of peptide drugs due to conformational changes, improved proteolytic stability and/or higher lipophilicity. Due to these characteristics N-methylated amino acids received increasing interest by the pharmaceutical industry.

Patchoulol Production with Metabolically Engineered .

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, has been engineered for the production of a number of value-added compounds including terpenoids.

Secretion of recombinant proteins from .

The microorganism is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years.

Improved fermentative production of the compatible solute ectoine by Corynebacterium glutamicum from glucose and alternative carbon sources.

The cyclic amino acid ectoine is a compatible solute serving as a protective substance against osmotic stress. Ectoine finds various applications due to its moisturizing effect. To avoid the disadvantages of the prevailing so-called "bacterial milking ectoine production process" caused by the high salt concentration, low salt fermentation strategies are sought after.

GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost.

Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development.

Streptavidin - a protein secreted by the filamentous bacterium Streptomyces avidinii - is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C.

Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli 'leaky mutant'.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed.

Applicability of Euglena gracilis for biorefineries demonstrated by the production of α-tocopherol and paramylon followed by anaerobic digestion.

In this study the use of Euglena gracilis biomass for α-tocopherol, paramylon and biogas production in a value-added chain was investigated. Therefore, we analyzed the dry cell weight and product concentrations at different growth phases during heterotrophic, photoheterotrophic and photoautotrophic cultivation in a low-cost minimal medium. Furthermore, the specific biogas yields for differently derived biomass with and without product recovery were investigated.

Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies.

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains.

Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength.

Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein.

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E.