Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture.

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.

Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing.

The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS.

Development and Characterization of an Enzyme Membrane Reactor for Fructo-Oligosaccharide Production.

Fructo-oligosaccharides (FOS) are linear fructans comprising 2-5 fructose units linked to a terminal glucose residue. They are widely used as food and feed additives due to their sweetness, low calorific value, and prebiotic properties. Here we describe the synthesis of FOS catalyzed by a cell-free crude enzyme solution containing recombinant fructosyltransferase (1-FFT) produced in the yeast Kluyveromyces lactis. During the enzyme catalysis, glucose accumulates as a by-product and eventually inhibits FOS production. We therefore used an enzyme membrane reactor (EMR) to achieve the continuous removal of glucose and the simultaneous replenishment of sucrose. We observed a loss of flux during the reaction with the characteristics of complete pore blocking, probably caused by a combination of proteins (enzyme molecules) and polysaccharides (FOS). Such complex fouling mechanisms must be overcome to achieve the efficient production of FOS using EMR systems.

Selection of High Producers From Combinatorial Libraries for the Production of Recombinant Proteins in Escherichia coli and Vibrio natriegens.

The optimization of recombinant protein production in bacteria is an important stage of process development, especially for difficult-to-express proteins that are particularly sensitive or recalcitrant. The optimal expression level must be neither too low, which would limit yields, nor too high, which would promote the formation of insoluble inclusion bodies. Expression can be optimized by testing different combinations of elements such as ribosome binding sites and N-terminal affinity tags, but the rate of protein synthesis is strongly dependent on mRNA secondary structures so the combined effects of these elements must be taken into account. This substantially increases the complexity of high-throughput expression screening. To address this limitation, we generated libraries of constructs systematically combining different ribosome binding sites, N-terminal affinity tags, and periplasmic translocation sequences representing two secretion pathways. Each construct also contained a green fluorescent protein (GFP) tag to allow the identification of high producers and a thrombin cleavage site enabling the removal of fusion tags. To achieve proof of principle, we generated libraries of 200 different combinations of elements for the expression of an antimicrobial peptide (AMPs), an antifungal peptide, and the enzyme urate oxidase (uricase) in Escherichia coli and Vibrio natriegens. High producers for all three difficult-to-express products were enriched by fluorescence-activated cell sorting. Our results indicated that the E. coli ssYahJ secretion signal is recognized in V. natriegens and efficiently mediates translocation to the periplasm. Our combinatorial library approach therefore allows the cross-species direct selection of high-producer clones for difficult-to-express proteins by systematically evaluating the combined impact of multiple construct elements.

Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli.

Peptides and proteins containing disulfide bonds can be produced in Escherichia coli by targeting the oxidizing periplasm, co-expressing isomerases or chaperons, refolding from inclusion bodies, or by using redox-engineered E. coli strains. Thus far, protein expression in glutathione reductase and thioredoxin reductase deficient (Δgor ΔtrxB) E. coli strains has required a complex medium. However, a chemically defined medium suitable for large-scale production would be preferable for industrial applications. Recently, we developed a minimal medium supplemented with iron (M9i) for high-density cultivation using E. coli Rosetta gami B(DE3)pLysS cells. Here we show that M9i is suitable for the production of insect metalloproteinase inhibitor (IMPI), which contains five disulfide bonds, in the same E. coli strain. We demonstrated the scalability of the new fed-batch process by combining the scale-up criteria of constant dissolved oxygen (DO) and matching volumetric power inputs (P/V) at the borders of the stirrer cascade. Process intensification was achieved by investigating production feed rates and different induction times. We improved product titers by ~200-fold compared to the standard process in complex medium while maintaining the activity of the IMPI protein. Our results show for the first time that it is possible to produce active proteins containing multiple disulfide bonds in a Δgor ΔtrxB E. coli strain using M9i medium. The success of scale-up and process intensification shows that the industrial production of complex recombinant proteins in such strains using chemically defined M9i minimal medium is feasible.

Downstream processing of Cry4AaCter-induced inclusion bodies containing insect-derived antimicrobial peptides produced in Escherichia coli.

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to the pull-down tag. The Cry4AaCter tag conserves the bioactivity of fusion proteins and thus allows the purification of simple AMPs and more complex AMPs stabilized by disulfide bonds. We developed a downstream process (DSP) for the purification of IBs containing the mutated Galleria mellonella insect metalloprotease inhibitor IMPI(I38V), which has a globular structure stabilized by five disulfide bonds. IMPI(I38V) is a potent inhibitor of the M4 metalloproteases used as virulence factors by several human pathogens. We used a single crossflow filtration for the washing and resolubilization of the Cry4AaCter-induced IBs and obtained bioactive IMPI(I38V) after tag removal. We achieved a 68-fold higher protein yield using our IB system compared to an alternative DSP approach in which a GST-fusion strategy was used to produce soluble IMPI(I38V). The Cry4AaCter-based process was transferable to gloverin (another G. mellonella AMP) and the visible marker green fluorescent protein, which accumulated in fluorescent IBs, confirming it is a broadly applicable strategy for the recovery of functional proteins.

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.