The aim of this thesis was to assess the functional expression of bacterial ligninases in mammalian cells, particularly in salivary cells, as a basic prerequisite for the potential generation of transgenic ruminants that can produce salivary ligninase with an enhanced utilization of high lignocellulosic fodders. Since mammalian and bacterial cells differ fundamentally in their cellular organization and functional characteristics, such as secretory pathways, codon-preference, and post-translational modifications of proteins (Mandad et al., 2018), it was not known if a functional expression of ligninases in mammalian cells is possible. Finally, one out of three tested ligninases, the DyP1 gene, could be shown to become secreted by the ParC10 salivary gland cell line and to exhibit an enzymatic degradation of Kraft lignin. Thus, a well characterized ligninase expression system was established, which may be used for further optimization and translation into agricultural use.
To achieve this milestone it was necessary to carefully design an expression construct for bacterial ligninases, to select suitable cell lines for expression testing, and to establish an assay for lignin degradation. The generic setup of the expression constructs was a polycistronic design which should facilitate expression and secretion of the ligninase gene, and which should also allow ligninase purification and direct vital recording of expression via a fluorescent tag. For this purposes, a synthetic expression cassette was projected, which included a mammalian promoter and a fusion construct of a mammalian secretory signal peptide with a codon-optimized and his-tagged ligninase followed by a 2A self cleaving peptide (2A) sequence and a Venus fluorophore with a NLS signal sequence. Thus, the polycistronic messenger RNA should be translated into a secreted his-tagged ligninase, whereas the Venus reporter should be directed into the nucleus.
The Venus protein can be exploited as a vital reporter for successful transfection, microscopic analysis, and for flow cytometric enrichment of expressing cells.
To achieve high efficient rates of transfected cells, I have initially optimized the square wave electroporation conditions for fibroblasts and salivary gland cells. In particular, the primary fibroblasts are considered as hard to transfect cells. The key parameter for electroporation is
“Maxwell-Wagner polarization”, which is an induced transmembrane voltage generated by an external electric field due to the variations in electrical properties of cell membrane, cytoplasm and external medium (Pavlin and Miklavčič, 2005). In a systematic test series of assessing amplitude of square wave pulse, pulse length, pulse number, electroporation buffer, plasmid amount, cell number and cuvette type the optimal pulse conditions found were a single 10 ms square-wave pulse of 400 volts in a 4 mm cuvette. Other critical conditions were i) the electroporation buffer ii) the electroporation temperature, iii) and the amount of plasmid DNA.
I observed that the electroporation buffer played the most critical role. Among the four tested electroporation buffers, Opti-MEM proved to be the best as it gave better transfection efficiency and superior cell viability in comparison to commercial Bio-Rad buffer, D10 medium and PBS.
Opti‐MEM is a serum‐reduced media and it contains insulin, transferrin, thymidine, hypoxanthine, and trace elements (Thermo Fisher). It also contains GlutaMAX supplement, which is a dipeptide, L-alanine-L-glutamine, and earlier studies have proven that polymers like poly-L-glutamate can increase efficacy and reduce toxicity of electroporation (Nicol et al., 2002). Thus, Opti-MEM ensures favorable Maxwell-Wagner polarization conditions, a prerequisite for better electroporation outcomes, this study confirms previous work that the electroporation buffer is a
critical factor for efficient transfections (Pavlin and Miklavčič, 2005). The details of this study are summarized in Manuscript I.
The generic DyP1 construct was synthetized by a commercial provider, unique restriction site flanking the Dyp1 gene allowed the exchange against the synthetic DyP2 and cotA genes. For stable expression, the constructs were then sub-cloned in a SB transposon vector (pT2-RMCE) (Mates et al. 2009, Garrels et al., 2016), which allowed the transposition into the genome by co-electroporation with a SB100x-transposase plasmid. It has been proven that the SB-mediated transposition favors euchromatic regions for integration and thus enables high and stable transcription rate (Mates et al., 2009; Garrels et al., 2011). In a second set of plasmids the ubiquitous CAGGS promoter was replaced by the 12 kbp PSP promoter with the aim of salivary gland-specific expression.
Promoters are generally located near the transcription start sites of genes, on the same strand and upstream on the DNA and are of variable length ((Dalton & Barton, 2014; Opabode and Akinyemiju, 2015) The ubiquitous CAGGS promoter was intended for high-level expression of the ligninases in cell type-independent manner (Sakaguchi et al., 2014). For the long term goal of a transgenic ruminant, it is important that the transgene expression is regulated very precisely in a tissue-specific manner (Tsubota et al., 2014). The use of tissue-specific expression in heterologous expression cassette minimizes the potential negative effects of a transgene on the physiology of transgenic animal (Zheng and Baum, 2008).
The commonly used ubiquitous promoters are cytomegalovirus (CMV) (Boshart, 1985) and SV40 (Neuhaus et al., 1986), human elongation factor-1 alpha (EF-1α) (Kim et al., 1990) and β-actin (Muller et al., 1990). The promoter activity can be further enhanced by addition of enhancer
elements upstream of the promoter, for example in the CAGGS promoter, the CMV immediate-early enhancer is fused to chicken β-actin promoter (Niwa et al., 1991). The CAGGS promoter is frequently been used to drive high-level gene expression in mammalian cells (Sakaguchi et al., 2014), and in transgenic animals (Garrels et al., 2011). Importantly, the ubiquitous expression of the CAGGS promoter has also been shown in SB-mediated genetically engineered cattle (Garrels et al., 2016).
Here, the CAGGS ubiquitous promoter was used to drive the secretion of ligninases in primary bovine fibroblast and ParC10 cells. The fluorescent microscopy revealed the expression of Venus protein in the nuclei, in addition, the Western blotting revealed DyP1 in cell culture medium.
However, the signal intensity Venus protein was stronger than DyP1 band in the Western blot assay. This might be due to differences in protein sizes, folding and post translational modifications, susceptibility to proteases, and different antibody affinities.
For the salivary tissue-specific expression, the parotid secretory protein promoter (PSP) was used. It is specifically expressed at high levels in the salivary glands and a 12-kb upstream promoter region of the gene necessary for tissue specific expression has been identified (Yin et al., 2006). Previously, the murine PSP promoter was used for expression of bacterial phytase in saliva of transgenic mice (Golovan et al., 2001a), and transgenic pigs (Golovan et al., 2001b). The PSP promoter was used for human nerve growth factor production in transgenic mice (Zeng et al., 2017), and co-expression of β-glucanase and xylanase in pigs (Zhang et al., 2018). Similarly, porcine PSP promoter was used for expressing beta-glucanase in saliva of mice (Guan et al., 2013) and pigs (Guan et al., 2017). Here, the murine PSP promoter was used to drive the tissue specific
expression of recombinant ligninases. The PSP promoter was found to be active only in salivary cells, but not in fibroblasts.
To direct the ligninase protein into the secretory pathway, the signal peptide (SP) sequence from human lysozyme was introduced in frame at the 5’ end of the cDNA. The nascent SP polypeptide is recognized by the signal recognition particle (SRP) and gets addressed to the endoplasmic reticulum (Saraogi and Shan, 2011) where it binds to SRP receptor and the SP is transferred to the translocon. Whilst bound to the translocon, translation is reinitiated and the protein passes through the ER membrane and into the lumen. In most cases the SP is recognised by a signal peptidase and is cleaved off to generate the mature protein that trafficks through the Golgi network before being secreted (Hegde and Bernstein, 2006). This translocation of secretory proteins into the lumen of the ER is a critical bottleneck within the secretory pathway (Zuccheli et al., 2016). The selection of appropriate signal peptide can have important consequences for the efficiency of protein secretion (Stern et al., 2011).
It is also established that many of the SPs are functionally interchangeable even between mammalian species despite their heterogeneity (Knappskog et al., 2007). This was attributed to three structurally conserved regions of a typical signal peptide, which are a N-terminal polar region (N-region), rich in positively charged amino acids, a central hydrophobic region (H-region) composed of about 7–8 hydrophobic amino acids, and a C-terminal region (C-region) that includes the SP cleavage site (Sakaguchi, 1997). Taking a cue from the above studies, I have used the lysozyme SP, since it is well characterized and naturally expressed in salivary gland cells (Lynge Pedersen and Belstrom, 2019). The Western blot results demonstrate the presence of DyP1 the cell culture medium, but not in the cell lysates, strong supporting the notion that the
DyP1 was indeed secreted. Nevertheless, it is worthwhile to test for other SPs in future studies to check the possibility for increasing the levels of target enzyme secretion in salivary gland cells (Hunter et al., 2018). The SP sequence can have dramatic effect on protein expression, in some case a four-fold enhancement of protein production was achieved (Kober et al., 2013).
Here, I utilized the Par-C10 salivary cell line (Quissel et al., 1998) for testing the proof of principle for mammalian ligninase production. Though both primary cells and immortalized cell lines can be used as salivary models, the primary cells are characterized by certain critical problems in vitro like tendency to de-differentiate when grown on plastic (Szlavik et al., 2008), committed to apoptosis when dissociated into single cell suspensions (Walsh et al., 1998), both of which can compromise viability. Immortalized cell lines offer many advantages like they are homogeneous, genetically identical, and easier to culture, and offer the possibility to extract large quantities of recombinant protein (Carter and Shieh, 2015).
This ParC10 cell line forms secretory granules, tight and intermediate junctions, desmosomes and microvilli (Quissel et al., 1998). When grown on plastic, ParC10 cells form monolayers of cuboidal cells with thick extracellular matrix at their base (Quissel et al., 1998). One feature of the cell line is absence of functional amylase expression when grown on plastic in reduced serum conditions (Zhu et al., 1998). The cells remained healthy post transfection and also withstood sorting procedures and pure population of cells were proliferated for up to 30 passages with robust DyP1 expression, indicating that the model was successful in testing our proof-of-concept.
Ligninases include a broad range of enzymes that can breakdown lignin with varying modes of action. Here, the functional expression of two classes of enzymes i.e., dye decolorizing peroxidases (from Thermobifida fusca (DyP1) and Paenibacillus JDR2 (DyP2) and laccase (cotA
laccase from Bacillus subtilis) were attempted. Dye decolorizing peroxidase (DyP) were chosen, since they belong to the class of heme peroxidases, which are ubiquitous in all domains of life (Chen and Li, 2016). They are also reported to have unique substrate specificity and catalytic properties, which offer great opportunities for biotechnological applications (Chen and Li, 2016).
DyP1 is well characterized in terms of its structure and activity; it is active at pH ranges of 3-10 and at temperatures of 20-40 0C (Rahmanpour et al., 2016). DyP2 gene from Paenibacillus JDR2 strain (Salvachua et al., 2015) was selected, because these bacteria are known to carry out lignolytic activity at alkaline pH (de Oliveira et al., 2009), the ideal pH for its functional expression in ruminant saliva. Similarly, cotA Laccase was chosen for the study considering its thermostability and alkaline activity (Wang et al., 2016).
Among the three enzymes assessed in mammalian cells, only DyP1 could be successfully expressed, whereas DyP2 and cotA could not be detected by Western blotting and after Ni-NTA purification. This can be due to either lower production or protein degradation by cellular proteases (Chakrabarti et al., 2016). Some of the reasons for low production are structural changes in recombinant gene, inefficient chromosomal integration, abnormal protein aggregation and folding, improper cleavage of signal peptide, and degradation by proteases (Chakrabarti et al., 2016). Interestingly, in both DyP2 and cotA transfected cells, the Venus gene encoded on the second cistron was expressed and could be readily detected in the cellular nuclei, suggesting that the polycistronic construct worked by that these ligninase were unstable.
For assessing the enzymatic activity of the DyP1 protein a new assay was established. Therefore the model compound Kraft lignin was added to the cell culture medium (without phenol red supplementation) and after 24 and 48 hours the cell culture medium was analyzed by
spectrophotometry for characteristic changes of the absorption spectra. At a concentration of 100ug/ml Kraft lignin the DyP1 expressing cells showed changes in the morphology characterized by rounding of cells and detachment from the surface, whereas this did not happen in wildtype, or DyP2 and cotA transfected cells. Most likely the phenomenon can be attributed as indirect evidence for the degradation of Kraft lignin. Expected degradation products are vanillin and eugenol, which cytotoxic for mammalian cells. The spectrophotometric analyses of the cell culture media confirmed a characteristic shift of the absorption, indicative for degradation products of Kraft lignin. The absorption shift was only found in culture media from the DyP1 expressing cells, but not in wildtype, or DyP2 and cotA transfected cells.
This ability of lignin breakdown is widespread in fungi, bacteria and archea (Janusz et al., 2017), but not in mammals or microbes of the rumen microbiome (Terry et al., 2019). Current progress in genetic engineering of livestock nowadays allows the introduction of exogenous genetic information into farm animal genomes to express entirely novel traits (Laible, 2009; Bosch et al., 2015). Depending on the species, practical applications of transgenic livestock include improved milk production and composition, increased growth rate and prolificacy, enhanced feed utilization efficiency, desirable carcass composition, and increased disease resistance (Wheeler, 2007). Hence, genetic modification of livestock with an aim to express certain desirable traits can offer a wide range of benefits to producers, consumers, society and environment. Up to now relative few applications were directed towards environmental benefits. One approach to this direction was the “Enviropig”, which aims at reducing the environmental impact of pork production by lowering the excretion of phosphorus in pig manure (Golovan et al., 2001).
Though recombinant production of functional bacterial enzymes in mammalian salivary system is not new, it is still essential to verify the proof of concept for every new intended applications (Kendig, 2015). The current study of establishing functional salivary ligninase expression as a fore runner for future transgenic ruminants with salivary ligninase activity is in broader sense an application directed towards a more environmentally sustainable farming.
In summary, this study demonstrate that mammalian salivary cells can be transduced to produce functional ligninase. In most of the developing and under developed countries, large quantities of crop residues such as cereal straws and stovers are generated, which are often burnt in heaps creating environmental pollution and associated problems. If genetically engineered cattle can efficiently utilize high lignoncellulosic by-products, their usefulness will be improved and production become more sustainable. The feasibility for SB-mediated transposition in bovine zygotes with the generation of genetically engineered cattle have been shown before (Garrels et al., 2016, Yum et al. 2016). Thus the here developed ligninase expression cassette within a SB transposon could be directly translated into animal generation and the characterization of the resulting cattle with respect to lignin digestion.
The greatest concern for this concept will be regulatory aspects with respect to transgenic animals. So far most of the genetic engineered livestock models intended an increase in production, manipulation of composition of livestock products, and disease resistance. But in the larger interests of environmental sustainability, if a concept is sufficiently convincing, then policy makers might ponder over the application of such a concept.
In most countries the commercialization of transgenic or genetically engineered animals is strictly regulated and only few products are approved. A prominent examples for an agricultural relevant
animal is the transgenically growth enhanced salmon (AquAdvantage salmon), which was approved by the United States (US) Food and Drug Administration (FDA) as safe food after a decade-long assessment, and is now commercialized in the USA and Canada (Benissa and Barbeiro, 2015). Biopharmaceutical medicaments from transgenic animals include recombinant anti-thrombin III (Atryn), human C1 esterase (Ruconest) and Sebelipase alfa (Kanuma). Atryn was the first biopharmaceutical product from a transgenic animal (goat milk) approved for therapeutic use in humans (Selokar and Kues, 2017). Atryn was approved in 2006 by the European Medicines Agency (EMA), and in 2009 by the FDA. Ruconest is a recombinant human C1 esterase inhibitor protein, produced in the milk gland of rabbits (van Veen et al., 2012; Selokar and Kues, 2017). In 2015, the FDA approved Kanuma (Sebelipase alfa) for the treatment of lysosomal acid lipase deficiency (Erwin, 2017). The recombinant Sebelipase alfa is isolated from the egg white of transgenic chickens. In addition, fluorescent zebrafishes (Glofish) are legally sold as pet toys in the USA, China and some other Asian countries.
In the EU, the definition of genetically modified organisms (GMO) include any organism (except human beings) in which genetic material has been altered in a way that does not occur naturally by mating or natural recombination (EC, 2001), and currently no approval of GMO animals for food production exist. Australia and New Zealand adopted in 1999 the standard 1.5.2 on “Food produced using gene technology”, as per which pre-market safety assessment, approval system for food produced using gene technology and mandatory labelling of food produced using gene technology were essential (Friedrichs et al., 2019). In case of India, there were rules passed in 1989 under the title ‘‘Rules for the manufacture, use, import, export and storage of hazardous microorganisms/genetically engineered organisms or cells’’ that covered the entire spectrum of
activities relating to research, development and use of GMOs and their products including new gene technologies (Chimata, 2019).
The recent technology of genome editing has revolutionized modern biology and already resulted in a wide range of agricultural applications (Carrol and Charo, 2015). The USA and Canada are regulating genome editing products according to a product-trigger, under which the relevant novelty of the trait in question was considered on a case-by-case basis, irrespective of the technology used to develop it (Friedrichs et al., 2019). As a consequence, genome edited plants and fungi, which carry only minute changes in their genome are not regulated in the USA and Canada. Several agricultural important nations, such as Argentina, Brazil, Australia and Russia follow this way. In contrast, the EU and New Zealand interpret genome edited organisms as GMO, and consequently apply the strict regulations as for transgenic organisms. Hence, though the future for genetic engineering and genome editing is challenging in terms of regulations and public perceptions, sustained efforts are needed from all the stakeholders to have a healthy science-based dialogue vis-à-vis considering societal and ethical concerns to strike a legitimate balance.
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