Author response: Novel transgenic pigs with enhanced growth and reduced environmental impact

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract In pig production, inefficient feed digestion causes excessive nutrients such as phosphorus and nitrogen to be released to the environment. To address the issue of environmental emissions, we established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphorus outputs in the transgenic pigs were reduced by 23.2–45.8%, and growth rate improved by 23.0% (gilts) and 24.4% (boars) compared with that of age-matched wild-type littermates under the same dietary treatment. The transgenic pigs showed an 11.5–14.5% improvement in feed conversion rate compared with the wild-type pigs. These findings indicate that the transgenic pigs are promising resources for improving feed efficiency and reducing environmental impact. https://doi.org/10.7554/eLife.34286.001 eLife digest The bodily waste that pigs produce contains high levels of chemicals that can damage the environment, such as nitrogen and phosphorus. For example, when excessive amounts of these two compounds make their way into the water, they can cause blue-green algae to grow too much, which asphyxiates other life in the water. Pigs produce a lot of nitrogen and phosphorus because they cannot efficiently digest their food. In particular, the animals lack the enzymes required to break down two types of molecules present in their feedstuff: phytates and non-starch polysaccharides (NSPs). Zhang, Li et al. take four microbial genes which code for the enzymes needed to digest NSPs and phytates, and they add these DNA sequences into the genomes of pigs. The animals then produce enzymes in their saliva that transform NSPs and phytates into molecules which can be used by their digestive system. The pigs thus get more energy from their food, and they grow faster and bigger. They also produce less nitrogen and phosphorus in their waste. Over 1.2 billion pigs are farmed each year, and they are the most economically important meat source in the world. Raising animals that can digest their food better would reduce the need for pig feed, increase productivity and reduce environmental pollution. However, discussions with policy makers and with the public will be necessary before these results can be adopted by the farming industry. https://doi.org/10.7554/eLife.34286.002 Introduction Annual global pig production is approximately 1.2 billion heads, with more than half produced in China (USDA, 2016). Grains are the main feedstuff of the pig industry; however, its production capacity in China and many other countries is insufficient. The livestock industry often allows animals to achieve maximal growth in order to fully utilize their economic outputs, yet inefficient feed digestion can cause serious nutrient emissions to the environment. Two nutrients that have received the most attention from environmental groups are nitrogen (N) and phosphorus (P) which are often supplied in excessive amounts in the diet in order to ensure maximal growth. In pig production, only 1/3 of feed N and P is metabolically utilized from cereal and soybean-based diets. The deposition rate of N is only 25–32% in grower-finisher pigs (Shirali et al., 2012). It has been reported that N excretion is up to 20 kg/sow/year and 25 kg/boar/year (DEFRA, 2007). Approximately 51% of N intake is excreted in urine, which is mainly from protein metabolism and underutilized amino acids and non-protein nitrogen (NPN) (Shirali et al., 2012). Fecal N excretion comes from undigested protein fractions and endogenous tissue losses such as digestive enzyme secretions and desquamation of intestinal cells, which accounts for 17% of the N intake (Dourmad et al., 1999). Only approximately 30% of P is retained in a grower-finisher pig on a cereal-soybean meal-based diet. In total, 70% of ingested P is excreted either through the feces or urine (Dourmad et al., 1999). The N and P from animal excreta pollute the water, soil, or air of intensive pig production sites (Osada et al., 2011; Philippe et al., 2011; Carter and Kim, 2013). Surface water becomes eutrophic following excessive P and N inputs, thereby causing overgrowth of blue-green algae and death of aquatic animals (Jongbloed and Lenis, 1998; Poulsen, 2000). Considering all these aspects, improving nutrient utilization in feed is of great significance to maximize feed grain utilization as well as for environmental conservation. Non-starch polysaccharides (NSPs) are primarily present in plant cell walls (McDougall et al., 1996; Sarkar et al., 2009). In cereal grains, arabinoxylans and β-glucans are found in the cell walls of the protein-rich aleurone layer and starchy endosperm and can act as a barrier to nutrient hydrolysis and absorption (Bacic and Stone, 1981). Similarly, the cell wall polysaccharides of soybean, canola seed, and peas may also be responsible for this nutrient-encapsulating effect (Omogbenigun et al., 2004). Therefore, NSPs are the main anti-nutrient factors of cereal and bran (Fangel et al., 2012; Sarkar et al., 2009). Due to a lack of endogenous NSP-degrading enzymes (NSPases), pigs are inherently incapable of digesting NSPs (Hooda et al., 2010), but can partially degrade this material through the action of the natural microbial community in their intestinal tract. High-P emission from monogastric animals such as pigs and poultry arises from their poor physiological ability to hydrolyze plant phytates, which account for up to 80% of P in common cereal grains, oil seed meals, and by-products (Ravindran et al., 1994). Phytates are negatively charged saturated cyclic acids that can bind to positively charged molecules in the diet such as minerals and protein, thereby reducing nutrient digestibility and increasing discharge of the unabsorbed nutrients to the environment (Dersjant-Li et al., 2015). Various methods have been employed to address the issues of inefficient utilization of feed nutrients in the pig industry. For example, dietary supplementation of phytate- or NSP-degrading enzymes has been proposed to reduce P or N emissions from pig farms (Kiarie et al., 2010; Zijlstra et al., 2010), as well as increasing pig body weight gain and feed conversion efficiency (Diebold et al., 2004; Willamil et al., 2012; Woyengo and Nyachoti, 2011). Recent advancements in genetic engineering and animal cloning technologies have facilitated in the establishment of genetically modified pigs with economically significant traits. Transgenic (TG) pig lines secreting salivary bacterial phytases have been generated previously. The P content of fecal matter from TG weaner and grower-finisher pigs fed on soybean meals was decreased by as much as 75% and 56%, respectively, compared to their non-TG counterparts. Endogenous salivary phytase significantly promoted the digestion of P from dietary phytates (Golovan et al., 2001). To our knowledge, no pig lines that express multi-NSP-degrading enzymes have been established to date. Here, we established stable transgenic pig lines that co-expressed NSP-degrading enzymes (β-glucanase and xylanase) and phytase in saliva. Multiple enzymes coordinately degrade NSPs and phytates in feed grains. We also report the grain digestibility, nutrient emission, growth performances, and feed conversion rate of the TG pigs compared with their wild-type littermates. Results Optimization and construction of a 2A-mediated salivary gland-specific multi-transgene Through characterization of multiple codon-optimized β-glucanase genes fused with the N-terminal porcine parotid secretory protein (PSP) signal peptide, we have determined that Bispora sp. MEY-1 endo-β-glucanase from (BG17A) and Bacillus licheniformisβ−1,3–1,4-glucanase (EG1314) exhibited optimal activity and stability in porcine cells, and the pH condition is compatible to that of the pig digestive tract (Zhang et al., 2015). We previously reported that the fused BG17A and EG1314, which was linked by a self-cleaving 2A peptide, had a broader optimal pH range and higher stability in an acidic environment than either of them alone (Zhang et al., 2015). After codon optimization and fusion with the pig PSP signal peptide, three xylanases (XYNB, XYL11, and XYF63 (also known as XYN11F63)) were transfected into PK15 cells and subjected to enzymatic activity assay. Among the three xylanases, XYNB presented the highest enzymatic activity (Figure 1—figure supplement 1A) and stability (Figure 1—figure supplement 1B). In addition, XYNB showed greater resistance to peptic and tryptic hydrolysis than the other two xylanases (Figure 1—figure supplement 1C–E). As for the two phytases, Citrobacter freundii APPA (CAPPA) only had two narrow peaks at the optimal pH levels of 2.5 and 5.0, respectively (Figure 1—figure supplement 2A), whereas Escherichia coli APPA (EAPPA) exhibited a broad optimal pH ranging from 1.5 to 5.0 (Figure 1—figure supplement 2B). EAPPA was more tolerant of pepsin and trypsin than CAPPA. There was almost no reduction in activity of EAPPA after a 2 hr pepsin treatment, whereas 52.2% of the biological activity was left for CAPPA after treatment (Figure 1—figure supplement 2C). When treated with trypsin alone, EAPPA and CAPPA retained 98.2% and 39.7% of their activity, respectively; when treated with trypsin +EDTA, EAPPA and CAPPA retained 31.8% and 13.7% of their activity, respectively (Figure 1—figure supplement 2D). Based on these results, two β-glucanases genes (bg17A and eg1314), a xylanase gene (xynB), and a phytase gene (eappA) showed better performance that the other candidate transgenes. A polycistronic cassette of fusion enzymes was constructed by head-to-tail ligation of four selected genes and flanked on the 3' end by an Hemagglutinin (HA) tag. The DNA sequences of the self-cleaving peptides E2A, T2A, and P2A were used as linkers between the coding DNA sequences of two neighboring enzymes (Figure 1—figure supplement 3A). Before construction of the final TG vector, the fusion enzyme sequences were ligated downstream of the CMV promoter, and the expression level and enzymatic activity of each fusion enzyme were measured in porcine cells. We were able to detect the expression of all the four active enzymes in the PK15 cells, although the expression level and enzymatic activity of each recombinant fusion enzyme was lower than its original monomeric counterpart (Figure 1—figure supplement 3B and C). The lower transfection efficiency of the large transgene construct likely accounted for the observed lower expression and enzymatic activity in cells. We then employed a mouse PSP promoter to replace the CMV promoter to control the salivary gland-specific expression of the fusion gene, and this expression cassette was inserted, together with a CMV promoter-driven neo-EGFP fusion gene, into the piggyBac transposon vector to form a TG vector, namely, pPB-mPSP-BgEgXyAp-neoGFP (Figure 1A). Figure 1 with 5 supplements see all Download asset Open asset Presence of the transgene in TG pigs. (A) DNA construct that was integrated into the pig genome for expression of the transgenic fusion enzyme in saliva. mPSP: Mouse parotid secretory protein promoter. BGH: Bovine growth hormone polyadenylation signal. Total length: 19,886 bp. (B) Expression of EGFP in the whole body of TG pigs. (C) Expression of EGFP in the heart, tongue, kidney, muscle, submandibular gland, spleen, lung, and liver of TG pigs. (D) Southern blot analysis of multi-enzyme transgene integration in TG pigs. 0.5 c, 1 c, 2 c, 3 c, and 5 c represent copy number of transgenic vector used as loading controls. The probe is shown in Figure 1A. Blank: Blank control (ddH2O). https://doi.org/10.7554/eLife.34286.003 Generation of TG pigs The resulting TG piggyBac transposon vector (pPB-mPSP-BgEgXyAp-neoGFP) and a piggyBac transposase expression vector (hyPBase) were co-transfected into the porcine fetal fibroblasts (PFFs) of a male Duroc pig. Transfected PFFs were selected with G418 for approximately two weeks, and the resulting EGFP-expressing cell colonies were pooled and identified by PCR for the presence of the transgene. Four colonies with great EGFP expression were used as donor cells for somatic cell nuclear transfer. A total of 4008 reconstructed embryos were generated and transferred to 16 recipient sows (Supplementary file 11). Thirty-three live and two stillborn cloned piglets were born, of which 25 founders were positive for transgene by PCR detection (Figure 1—figure supplement 4A and B). Bright green fluorescence signals were observed in hoof, tongue, heart, muscle, and submandibular gland (Figure 1B and C). Among the 25 TG founders, five piglets (601, 603, 701, 705, and 709) harbored the fragments of the ampicillin-resistance gene of the transgene vector (Figure 1—figure supplement 4A), implying the occurrence of a random, but not transposon-mediated transgene integration into host cell genome. The other 20 piglets harbored the intact transgene expression cassette (total length: 19,886 bp). Southern blotting, quantitative PCR, inverse PCR, and sequencing results further demonstrated that 19 piglets carried a single copy of the transgene (Figure 1D; Figure 1—figure supplement 4C; Figure 1—figure supplement 5; Supplementary file 1), of which two carried a single copy of the transgene that was inserted into intron 1 of Legumain (line 1) (Figure 1—figure supplement 4C; Figure 1—figure supplement 5; Supplementary file 1), 17 carried a single copy of the transgene that was integrated into intron 5 of CEP112 (line 2, of which eight piglets survived) (Figure 1D; Figure 1—figure supplement 5; Supplementary file 1). One (708) carried three copies of the transgene, and the integration site was the intergenic region between LOC100525528 and CXCL2 (Figure 1D; Supplementary file 1). There were a total of 16 piglets survived to weaning. Thirteen of them are positive for transgene, including nine piggyBac-mediated (one of line 1 and 8 of line 2) and four randomly integrated transgenic pigs. Eight of the transgenic pigs survived to sexual maturity. RT-PCR analysis indicated that the BgEgXyAp, bg17, eg1314, xynB, and eappA transgenes were unambiguously expressed in the parotid, submandibular, and sublingual glands, whereas these were undetectable in the other tissues of the TG founders such as the lungs, heart, liver, stomach, spleen, kidney, duodenum, colon, and muscle (Figure 2A; Figure 2—figure supplement 1A). Quantitative PCR analysis indicated that the highest BgEgXyAp transgene expression levels were observed in the parotid gland, followed by the submandibular and sublingual glands, and trace or undetectable level were observed in the other tissues of the TG founders (Figure 2—figure supplement 1B). Ectopic expression was not observed in this study. Western blot analysis demonstrated the expression of β-glucanase, xylanase, and phytase in the saliva of the TG founders (Figure 2B). During the feeding period, the TG pigs (line 1 and line 2) produced 0.3–2.3 U/mg of β-glucanase, 0.6–2.4 U/mg of xylanase, and 0.5–5.7 U/mg of phytase in the saliva (Figure 2C–E). The total salivary protein concentrations of the TG and WT pigs are shown in Figure 2F. Figure 2 with 2 supplements see all Download asset Open asset Expression and enzymatic activity of transgenes in TG pigs. (A) RT-PCR assay for mRNA expression profiles of transgenes in different tissues. Forward primers and reverse primer are bound to the bg17A gene and eappA gene, respectively (arrows are shown in Figure 1A). Mock: Blank control (ddH2O). (B) Western blotting assay demonstrating the expression of BG17A, XYNB, and EAPPA in the saliva of TG pigs. Saliva samples were either incubated with PNGase F (+) or mock (-)-treated prior to western blotting to analyze the glycosylation status of the transgenic enzymes. PNGase F: Peptide N-glycosidase F. (C–E) Salivary β-glucanase, xylanase, and phytase activity assays of the TG pigs. (F) Concentration of total salivary protein of the TG and WT pigs. C1, C2: Age- and body weight-matched WT pigs. The data presented in the figure (C-E) can be found in Figure 2—source datas 1–4. https://doi.org/10.7554/eLife.34286.009 Figure 2—source data 1 Salivary β-glucanase activity assays of TG pigs. https://doi.org/10.7554/eLife.34286.012 Download elife-34286-fig2-data1-v1.xlsx Figure 2—source data 2 Salivary xylanase activity assays of TG pigs. https://doi.org/10.7554/eLife.34286.013 Download elife-34286-fig2-data2-v1.xlsx Figure 2—source data 3 Salivary phytase activity assays of TG pigs. https://doi.org/10.7554/eLife.34286.014 Download elife-34286-fig2-data3-v1.xlsx Figure 2—source data 4 Concentration of total salivary protein of TG and WT pigs. https://doi.org/10.7554/eLife.34286.015 Download elife-34286-fig2-data4-v1.xlsx Pigs of TG line two that harbored a single-copy transgene within CEP112 intron were used in growth trials and feed evaluations. The TG line two pigs were crossed with WT Duroc pigs, which generated 116 F1 progeny, of which 57 tested positive for transgene. Furthermore, 404 of the F2 progeny were sired, of which 231 were positive for transgene. Measurement of enzyme production in TG pigs To understand the effect of the three enzymes of the TG pigs on nutrient digestion, we investigated the pattern of salivary secretion and enzyme production in the TG pigs. Saliva was collected from the unilateral parotid glands of the TG pigs and analyzed in terms of enzyme yield. The average β-glucanase, xylanase, and phytase yields were 2,331.8, 2,413.4, and 2,935.2 U per kilogram meal, respectively, in grower pigs, and 920.8, 939.0, and 1,042.2 U per kilogram meal, respectively, in finisher pigs (Table 1). The volume of saliva collected from the parotid gland of the finisher pigs was significantly lower compared to that of the grower pigs, which may be attributable to the shorter feeding time (Ft) in finisher pigs. Furthermore, saliva and enzyme production at different time points were evaluated. The results show that the pigs only secreted saliva from parotid gland during Ft. At the other time points, including 10 min before or after feeding (Bf or Af), insignificant amount of saliva was secreted (Figure 2—figure supplement 2A). The enzymes expressed by the transgene showed high enzymatic activity at Bf, Ft, and Af. At rest time (Rt), the saliva collected from either parotid or mouth showed reduced enzymatic activities. Of note, a significantly lower enzyme activity was observed in the saliva samples collected at Rt (Figure 2—figure supplement 2B). Table 1 Salivary secretion and the transgene enzyme activities from the unilateral parotid gland of transgenic (TG) pigs (Line2) during the grower (92–96 days old; estimated body weight: 42–45 kg) and finisher (159–191 days old; estimated body weight: 100–115 kg) phases of growth. https://doi.org/10.7554/eLife.34286.016 ItemGrowth stagesPooled SEMP valuesGrowerFinisherSaliva secreted * Saliva secretion rate (mL/min·pig)8.4816.97**0.25<0.0001Saliva secretion volume (mL/kg diet consumed)407.66**151.705.33<0.0001Enzymes activity secreted (U/mL saliva)†β-glucanase5.726.07**0.05<0.0001Xylanase5.926.19**0.050.0005Phytase7.26.870.140.1033Enzyme activity secreted (U/kg diet consumed)†β-glucanase2,331.84**920.8230.7<0.0001Xylanase2,413.38**939.0331.73<0.0001Phytase2,935.19**1,042.1938.29<0.0001 *Saliva samples were collected daily from two transgenic growing pigs and two transgenic finishing pigs at 9:00 and 16:00, respectively, the are expressed as the and pooled 16 and and pooled and indicate significant between the grower and the finisher phases within the same Table data 1 Salivary secretion from the unilateral parotid gland in the growing transgenic (TG) pigs and pigs during the grower (92–96 days old; estimated body weight of 42–45 Download Table data 2 Salivary secretion from the unilateral parotid gland in the transgenic (TG) pigs during the finisher (159–191 days old; estimated body weight of 100–115 Download Table data 3 The transgene enzyme activities from the unilateral parotid gland in the transgenic (TG) pigs during the grower (92–96 days old; estimated body weight of 42–45 kg) and the finisher phases (159–191 days old; estimated body weight of 100–115 Download feed utilization and reduced nutrient emission in TG founders The grower-finisher TG pigs to kg) were fed or (Supplementary file 2) to the of a salivary of β-glucanase, xylanase, and phytase on feed The diet contains a level of NSPs and total P with a high of phytates and the diet contains a high of NSPs and total P with For each TG pigs and age-matched and weight-matched non-TG Duroc were and were fed the same diet with multi-enzyme of β-glucanase, xylanase, and phytase After dietary treatment, nutrient digestion the groups was measured and For the total tract digestibility of matter and significantly in TG pigs compared with that of WT pigs (Figure 3A). The fecal outputs of N and to by feed, were significantly decreased in the TG pigs compared with that of the WT pigs (Figure Fecal N and P excretion were decreased by and respectively, with the and and respectively, with the diet. A significant reduction in total P and was also observed in the TG pigs with diets. all tested of the TG pigs showed improvement compared with that of the pigs that were fed the same with multi-enzyme although the were not significant groups (Supplementary file 3 and Supplementary file Figure 3 Download asset Open asset of the total tract nutrient digestibility and fecal nutrient of their dietary between transgenic (TG) grower pigs (Line2) and their wild-type littermates fed on and soybean and and and soybean with and feed enzymes. (A) of the total tract nutrient digestibility of matter protein phosphorus and (B) of fecal and WT grower pigs fed on the and with an optimal of β-glucanase, xylanase, and are expressed as the on the with different significantly The source data are presented in Figure datas Figure data 1 of the total tract nutrient digestibility of and Download Figure data 2 of the total tract nutrient digestibility between transgenic (TG) grower pigs and their wild-type littermates fed and soybean Download Figure data 3 of the total tract nutrient digestibility between transgenic (TG) grower pigs and their wild-type littermates fed and soybean Download Figure data 4 of fecal fecal P and fecal Download Figure data 5 of fecal fecal P and fecal by transgenic (TG) grower pigs and their wild-type littermates fed and soybean with and feed enzymes. Download Figure data of fecal fecal P and fecal by transgenic (TG) grower pigs and their wild-type littermates fed and soybean with and feed enzymes. Download The of the TG pigs fed on a diet in Supplementary file 5 a N level and high of phytates were activity in the TG pigs was lower than the WT littermates. P and levels of the TG pigs were greater than that of the WT littermates. The levels of the TG pigs were higher than the WT littermates. in and total protein concentrations between TG and WT pigs were observed (Table Table 2 of the in the F1 transgenic (TG) grower (Line2) and the wild-type kg) fed on the diet in Supplementary file SEMP phosphorus N protein were collected at the end of the indicate significant at between TG and WT pigs within the same Table data 1 of the in the F1 transgenic (TG) grower and the wild-type kg) fed the non-starch diet. Download growth performance in TG pigs To the growth eight F1 TG pigs and 17 WT littermates were fed a diet (Supplementary file during the growing from to was The TG pigs exhibited a higher average daily gain rate and lower feed conversion rate than the WT pigs during this (Supplementary file performance of the F2 TG pigs was also A total of F2 TG pigs and WT littermates were together and fed the same shown in Supplementary file In this TG showed average daily feed intake compared to the WT that were fed the same (Figure results were observed in the improved and lower were observed in the TG and compared to the WT during the feeding (Figure and C). It an average of days for TG to grow from to whereas the WT needed days of feeding on the same diets. results were observed in the It days for the TG to grow from to whereas the WT required days (Figure the TG pigs showed a higher than the WT pigs the same grower and finisher phases to but more body weight daily than the WT pigs the same grower and finisher phases to The time to was by days to The decreased by 11.5–14.5% during the grower and finisher Figure 4 Download

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