Advances in Editing Silkworms (Bombyx mori) Genome by Using the CRISPR-Cas System

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) represents a powerful genome editing technology that revolutionized in a short period of time numerous natural sciences branches. Therefore, extraordinary progress was made in various fields, such as entomology or biotechnology. Bombyx mori is one of the most important insects, not only for the sericulture industry, but for numerous scientific areas. The silkworms play a key role as a model organism, but also as a bioreactor for the recombinant protein production. Nowadays, the CRISPR-Cas genome editing system is frequently used in order to perform gene analyses, to increase the resistance against certain pathogens or as an imaging tool in B. mori. Here, we provide an overview of various studies that made use of CRISPR-Cas for B. mori genome editing, with a focus on emphasizing the high applicability of this system in entomology and biological sciences.

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.

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Development of an Autoinducible Plasmid for Recombinant Protein Production

Background: The production of recombinant proteins in E. coli involves such factors as host strains, expression vectors, culture media, and induction methods. The typical procedure to produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable vector to construct an overexpression plasmid, (2) transformation of a strain specialized for protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium and induction of the protein production at a right moment, and (4) further growth to get the maximum yield. There are hurdles involved in each of these steps, and researchers have developed many materials or methods, which often require special recipes or procedures. Objective: To eliminate the special requirements for the recombinant protein production by using readily available materials. Also to save time and effort in the routine protein production work. Method: We started with a vector capable of producing a target protein fused to the C-terminus of the maltose binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase) was replaced with sacB. We chose 3 stationary phase promoters, and made hybrids of them by mixing halves from each one. The T5 promoter was replaced with these stationary phase promoters or their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST genes were inserted in place of sacB, and their production yields were compared with the original plasmid in the conventional way of expression. Results: We constructed an expression plasmid with an autoinducible promoter working in a host that was not specially designed for protein production and in a TB medium which did not contain any secret ingredient, nor was difficult to prepare unlike Studier’s defined medium. This plasmid also contains a color indicator which turns red when protein production is successful. We tested our system with the maltose binding protein (MBP) and the glutathione S-transferase (GST), and showed that both proteins were produced to a level comparable to what the commercial medium and/or the specialized strain yielded. Conclusion: We developed a plasmid equipped with an autoinducible promoter, a hybrid of the two promoters which were activated at the stationary phase. This plasmid does not need a special E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color, which can be used as an indicator of a successful production of the target protein and as a predictive measure of the amount of the produced target protein. We speculate that this plasmid will have its greatest advantage when growing cells at low temperatures which would inevitably take a long time.

Genome scale modeling of the protein secretory pathway reveals novel targets for improved recombinant protein production in yeast

Eukaryal cells are used for the production of many recombinant pharmaceutical proteins, including several of the current top-selling products. The protein secretory pathway in eukaryal cells is complex and involves many different processes such as post-translational modifications, translocation, and folding. Furthermore, recombinant protein production competes with native secretory proteins for the limited energy and proteome resources allocated to the protein secretory pathway. Due to the complexity of this pathway, improvement through metabolic engineering has traditionally been relatively ad-hoc; and considering the industrial importance of this pathway, there is a need for more systematic approaches for novel design principles. Here, we present the first proteome-constrained genome-scale protein secretory model of a eukaryal cell, namely for the yeast Saccharomyces cerevisiae (pcSecYeast). The model contains all key processes of this pathway, i.e., protein translation, modification, and degradation coupled with metabolism. The model can capture delicate phenotypic changes such as the switch in the use of specific glucose transporters in response to changing extracellular glucose concentration. Furthermore, the model can also simulate the effects of protein misfolding on cellular growth, suggesting that retro-translocation of misfolded proteins contributes to protein retention in the Endoplasmic reticulum (ER). We used pcSecYeast to simulate various recombinant proteins production and identified overexpression targets for different recombinant proteins overproduction. We experimentally validated many of the predicted targets for α-amylase production in this study, and the results show that the secretory pathways have more limited capacity than metabolism in terms of protein secretion.

Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann’s ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.