Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

Engineering Saccharomyces Cerevisiae for the Production and Secretion of Affibody Molecules.

BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to address if improvements in terms of yield, ease of production and if purification advantages can be identified. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, showed to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind human HER3 as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching high yields and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.

First time β-farnesene production by the versatile bacterium Cupriavidus necator

Background Terpenes are remarkably diverse natural structures, which can be formed via two different pathways leading to two common intermediates. Among those, sesquiterpenes represent a variety of industrially relevant products. One important industrially produced product is β-farnesene as a precursor for a jet fuel additive. So far, microbial terpene production has been mostly limited to known production hosts, which are only able to grow on heterotrophic substrates. Results In this paper, we for the first time describe β-farnesene production by the versatile bacterial host Cupriavidus necator on fructose, which is known to grow hetero- and autotrophically and even in bioelectrochemical systems. We were able to show a growth-dependent production of β-farnesene by expressing the β-farnesene synthase from Artemisia annua in C. necator H16 PHB-4. Additionally, we performed a scale-up in a parallel reactor system with production titers of 26.3 ± 1.3 µM β-farnesene with a fed-batch process. Conclusions The β-farnesene production titers reported in this paper are not in the same range as titers published with known heterotrophic producers E. coli or S. cerevisiae. However, this proof-of-principle study with C. necator as production host opens new synthesis routes toward a sustainable economy and leaves room for further optimizations, which have been already performed with the known production strains.

Microparticles and Nanoparticles from Plants—The Benefits of Bioencapsulation

The efficacy of drugs and vaccines depends on their stability and ability to interact with their targets in vivo. Many drugs benefit from encapsulation, which protects them from harsh conditions and allows targeted delivery and controlled release. Although many encapsulation methods are inexpensive, such as the formulation of tablets for oral delivery, others require complex procedures that add significantly to production costs and require low-temperature transport and storage, making them inaccessible in developing countries. In this review we consider the benefits of encapsulation technologies based on plants. Plant-derived biopolymers such as starch and the maize storage protein zein are already used as protective coatings, but plant cells used as production host provide natural in vivo bioencapsulation that survives passage through the stomach and releases drugs in the intestine, due to the presence of microbes that can digest the cell wall. Proteins can also be encapsulated in subcellular compartments such as protein bodies, which ensure stability and activity while often conferring additional immunomodulatory effects. Finally, we consider the incorporation of drugs and vaccines into plant-derived nanoparticles assembled from the components of viruses. These are extremely versatile, allowing the display of epitopes and targeting peptides as well as carrying cargoes of drugs and imaging molecules.

Metabolic engineering of Vibrio natriegens

Vibrio natriegens is emerging as a promising host for biotechnology which is basically due to the remarkable intrinsic properties such as the exceptionally high growth and substrate consumption rates. The facultatively anaerobic marine bacterium possesses a versatile metabolism, is able to utilize a variety of substrates as carbon and energy sources and is easy to handle in the lab. These features initiated the rapid development of genetic tools and resulted in extensive engineering of production strains in the past years. Although recent examples illustrate the potential of V. natriegens for biotechnology, a comprehensive understanding of the metabolism and its regulation is still lacking but essential to exploit the full potential of this bacterium. In this review, we summarize the current knowledge on the physiological traits and the genomic organization, provide an overview of the available genetic engineering tools and recent advances in metabolic engineering of V. natriegens. Finally, we discuss the obstacles which have to be overcome in order to establish V. natriegens as industrial production host.

Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production

Background Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce. Results We subjected Saccharomyces cerevisiae, a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l−1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula. When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l−1. Conclusions In this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.

Optimization of steam explosion parameters for improved biotechnological use of wheat straw

Using lignocellulosic raw materials as substrate for biotechnological applications has been a focus of research during the last two decades. They contain sugars, which can be used in industrial fermentation processes, in from of polysaccharides (cellulose, hemicellulose). Wheat straw, one representative of lignocellulosic materials, is sustainably and abundantly available, especially in Europe and North America. However, wheat straw, just like any other lignocellulosic material, needs to be pretreated in one way or the other in order to generate sufficient quantities of monosaccharides. One widely used pretreatment for lignocellulosic material is steam explosion combined with enzymatic hydrolysis. In this study, the effects of steam exploding wheat straw in combination with water are presented. By impregnation with water, saccharide yields from subsequent enzymatic hydrolysis increased from 18.8 to 22.6 g L−1 for glucose and 13.8 to 16.4 g L−1 for xylose, respectively. Moreover, the basic steam explosion parameters residence time and temperature were optimized in ranges from 5 to 20 min and 180–200 °C. This further optimization increased the maximum saccharide yield to 41.2 g L−1 for glucose (200 °C, 15 min) and 18.9 g L−1 for xylose (190 °C, 10 min). Finally, the growth of the intensively investigated biotechnological production host Yarrowia lipolytica on hydrolysates derived from different steam explosion parameters was evaluated. Y. lipolytica grew well in media containing up to 90% wheat straw hydrolysate as sole carbon source, demonstrating the potential as substrate for biotechnological processes.