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Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

ABSTRACT: Phytase is one of the most effective feed additives to increase the availability of phosphorus and minerals by catalyzing the hydrolysis of phytic acid. A modified appA gene (mappA) was transformed into soybean (Glycine max) under the control of a seed-specific promoter from common bean (Phaselous vulgaris). The soybean recombinant phytase showed optimal activity at pH 4.5 and 70?°C. A slight increase in enzyme activity occurred when the recombinant enzyme was pre-incubated with n-hexane. In addition, the phytase activity from our transgenic soybean does not reduce even after 2?hours of extraction with n-hexane at 55~65?°C. In conclusion, the oil extraction process using n-hexane does not inactivate the phytase expressed in the mAppA transgenic soybean, and the meal derived from the transgenic soybean processing can be used as feed supplement to livestock.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC6779883 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

Zhao Yu Y Zhu Lixia L Lin Chaoyang C Shen Zhicheng Z Xu Chao C

Scientific reports 20191007 1

Phytase is one of the most effective feed additives to increase the availability of phosphorus and minerals by catalyzing the hydrolysis of phytic acid. A modified appA gene (mappA) was transformed into soybean (Glycine max) under the control of a seed-specific promoter from common bean (Phaselous vulgaris). The soybean recombinant phytase showed optimal activity at pH 4.5 and 70 °C. A slight increase in enzyme activity occurred when the recombinant enzyme was pre-incubated with n-hexane. In add ...[more]

PMID: 31591515

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Project description:BackgroundIn plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate. However, phytate is poorly digested by monogastric animals such as poultry, swine and fish, as they lack the hydrolytic enzyme phytase; hence it is regarded as a nutritionally inactive compound from a phosphate bioavailability point of view. In addition, it also chelates important dietary minerals and essential amino acids. Therefore, dietary supplementation with bioavailable phosphate and exogenous phytases are required to achieve optimal animal growth. In order to simplify the obtaining and application processes, we developed a phytase expressing cell-wall deficient Chlamydomonas reinhardtii strain.ResultsIn this work, we developed a transgenic microalgae expressing a fungal phytase to be used as a food supplement for monogastric animals. A codon optimized Aspergillus niger PhyA E228K phytase (mE228K) with improved performance at pH 3.5 was transformed into the plastid genome of Chlamydomonas reinhardtii in order to achieve optimal expression. We engineered a plastid-specific construction harboring the mE228K gene, which allowed us to obtain high expression level lines with measurable in vitro phytase activity. Both wild-type and cell-wall deficient strains were selected, as the latter is a suitable model for animal digestion. The enzymatic activity of the mE228K expressing lines were approximately 5 phytase units per gram of dry biomass at pH 3.5 and 37 °C, similar to physiological conditions and economically competitive for use in commercial activities.ConclusionsA reference basis for the future biotechnological application of microalgae is provided in this work. A cell-wall deficient transgenic microalgae with phytase activity at gastrointestinal pH and temperature and suitable for pellet formation was developed. Moreover, the associated microalgae biomass costs of this strain would be between US$5 and US$60 per ton of feedstuff, similar to the US$2 per ton of feedstuffs of commercially available phytases. Our data provide evidence of phytate-hydrolyzing microalgae biomass for use as a food additive without the need for protein purification.

Project description:BackgroundGenetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood.ResultsTo investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes.ConclusionsThis study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Dataset Information

Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

Publications

Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

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