Alternative strategy enables automation of up- and downstream processes for recombinant production of an antimicrobial peptide in E. coli

Standard production processes for antimicrobial peptides (amps) comprise of batch cultivations in upstream and chromatographic purification in down-stream. Although chromatography in most variations is limited in scale-up and one of the most expensive steps in the production chain it still remains the industrial gold standard in downstream processing. To overcome this bottleneck in the production chain, alternative column-free purification strategies with aggregating tags are a promising approach. Such a process can be automated and scaled-up in consent with reasonable expenses. However, new processes involving alternative downstream strategies have to be developed starting from scratch. In regards to vector design it must be considered whether the product is intended to be expressed in a soluble or insoluble form, in which cell compartment it shall be synthesized or what kind of plasmid features are needed for the realization of an automated downstream process. In this study the soluble production of an amp with several structural disulfide bonds was conducted in an E. coli strain with an oxidizing cytoplasm. To generate the expression vector a unique combination of plasmid features was assembled by Golden Gate cloning to enable an automated downstream procedure with aggregating tags for column-free purification. The junction of a thioredoxin-tag (trxA) with elastin-like-polypeptides (ELPs) and self-splicing inteins allows the release of the protein from the cytoplasm into the extracellular space and the separation from host cell proteins (HCP) in the bioreactor vessel. The trxA-tag acts as a solubility enhancer to the product and facilitates the release into the extracellular space via simple osmotic shock procedure. Reversible, temperature dependent phase transition of the ELPs enables purification of the desired product from impurities such as HCP and, after cleavage by self-splicing inteins, to separate the amp from the tags itself. Please click Additional Files below to see the full abstract

Intended insoluble expression of recombinant protein with a pull-down tag in E. coli for simplifying product purification and increasing yield

The markets for recombinant proteins are growing and for most applications there is a demand for soluble recombinant protein. However, recombinant protein production in Escherichia coli often triggers insoluble protein production. Insoluble protein is either degraded or accumulates in solid inclusion bodies with high specificity. This means there is a high purity grade of the recombinant protein in inclusion bodies that can reach up to 90%. Moreover, protein in inclusion bodies is hardly degraded. However, the protein is mostly not accessible in an easy way and has to be denatured to resolubilize the inclusion body structure. The native protein structure has to be reestablished in an empirical manner via refolding procedures which account for high yield losses. There are two general possibilities to avoid the aforementioned struggles of insoluble protein production. First, the target protein can be designed by targeted mutations that can increase its solubility or by fusing the target protein to tags that convey solubility. There is a high chance to get a bioactive protein, but the advantage of high initial product purity and product stability is lost. Second, the protein can be designed with certain tags that convey accumulation in inclusion bodies under known molecular circumstances. These tags can also enable an easy resolubilization without denaturants. Thus, it is also possible to obtain bioactive protein and one profits by the high initial product purity and stability in inclusion bodies. One tag of special interest is the 158 amino acid Cry4AaCter-tag from Bacillus thuringiensis israelensis [1, 2]. It pulls down the protein of interest into inclusion bodies and resolubilization is easily achieved by transfer to basic pH milieu. This study aims for the recombinant production of an antimicrobial peptide (amp) in inclusion bodies in E. coli using the Cry4AaCter-tag. The applied model amp is the 70 amino acids Galleria mellonella insect metalloproteinase inhibitor (IMPI), which contains five disulfide bonds [3]. The production of this disulfide-rich peptide in E. coli was accomplished applying a genetically engineered strain with a non-reducing cytoplasmic milieu. High yields of fusion protein were already obtained at lab-scale in shaking flasks, which were almost completely resolubilized by simple change into basic pH milieu. The stability of IMPI enabled acid precipitation after tag cleavage, because IMPI stays soluble in presence of trichloroacetic acid while the cleaved tag and the protease precipitate. The activity of the product was confirmed in a fluorescence-based assay where IMPI inhibits a metalloprotease. An eukaryotic potential drug candidate protein was produced in E. coli in its bioactive form with high purity via this inclusion body-based production strategy with rendering denaturants, refolding procedures and chromatography steps obsolete. This system holds great promise for increasing demands of recombinant protein and will be further investigated with other proteins of interest. [1] Hayashi, M., Iwamoto, S., Sato, S., Sudo, S., Takagi, M., Sakai, H. u. Hayakawa, T.: Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli. Protein Expression and Purification 88 (2013) 2, S. 230–234 [2] Hayakawa, T., Sato, S., Iwamoto, S., Sudo, S., Sakamoto, Y., Yamashita, T., Uchida, M., Matsushima, K., Kashino, Y. u. Sakai, H.: Novel strategy for protein production using a peptide tag derived from Bacillus thuringiensis Cry4Aa. The FEBS journal 277 (2010) 13, S. 2883–2891 [3] Wedde, M., Weise, C., Kopacek, P., Franke, P. u. Vilcinskas, A.: Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella. European Journal of Biochemistry 255 (1998) 3, S. 535–54

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2±151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8±8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.Jacobs University Bremen gGmbH; European Union (PGSYS/ETB-2008-44

Ground Water Well Design and PFAS Treatment

Project Description: Group E2 is working with AKM Consulting Engineers to design a groundwater well to meet an increase in water demand for South Montebello Irrigation District. In addition, a Perfluoroalkyl (PFAS) treatment, will be added on-site to target and remove PROA and PFOs, which were found to be in the water supply during feasibility testing of the well site determination. Industrial Advisor: Zeki Kayiran, P.E. AKM Consulting Enginee

Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: A comparative study

WOS: 000303188100004A comparative evaluation of three Aspergillus species according to their pectinase production in solid-state fermentation was performed. Solid-state fermentation offers several potential advantages for enzyme production by fungal strains. Utilization of agricultural by-products as low-cost substrates for microbial enzyme production resulted in an economical and promising process. The pectinolytic enzyme activities of two Aspergillus sojae strains were compared to a known producer, Aspergillus niger IMI 91881, and to A. sojae ATCC 20235, which was re-classified as Aspergillus oryzae. Evaluation of polymethylgalacturonase and polygalacturonase activity was performed as well as exo- vs. endo-enzyme activity in the crude pectinase enzyme-complex of the mentioned strains. Furthermore, a plate diffusion assay was applied to determine the presence and action of proteases in the crude extracts. A. sojae ATCC 20235 with highest polymethylgalacturonase activity and highest polygalacturonase activity both exo- and endo-enzyme activity, is a promising candidate for industrial pectinase production, a group of enzymes with high commercial value, in solid-state fermentation processes. Beside the enzymatic assays a protein profile of each strain is given by SDS-PAGE electrophoresis and in addition species-specific zymograms for pectinolytic enzymes were observed, revealing the differences in protein pattern of the A. sojae strains to the re-classified A. oryzae. (C) 2011 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.Jacobs University Bremen gGmbH [PGSYS/ETB-2008-41]; Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [107O602]Financial support of Jacobs University Bremen gGmbH through the project PGSYS/ETB-2008-41 and Scientific and Technological Research Council of Turkey (TUBITAK) through the project 107O602 is gratefully acknowledged