Overexpression of a Fungal β-Mannanase from Bispora sp. MEY-1 in Maize Seeds and Enzyme Characterization

Abstract

BackgroundMannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.Methodology/Principal FindingsThe man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.Conclusion/SignificanceThis study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Transgenic cereals: Current status and future prospects

“…The value of such low-phytate maize products has been recently confirmed in feeding trials with poultry (Gao et al, 2012;Ma et al, 2013;Wang et al, 2013e) and pigs (Li et al, 2013d). A similar benefit may derive from GM maize expressing a fungal β-mannanase from Bispora (Xu et al, 2013b).…”

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

“…The preparation of polyclonal antibody of Aga-F75 in rabbits (6 pounds and greater) was conducted in the Laboratory of Animal Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Beijing, China) following the rules and protocols approved by the Experimental Animal Ethics Committee of Chinese Academy of Sciences. The purification of polyclonal antibody and the procedures of Western blot were carried out as described previously [ 17 ]. The bands were then excised from the gel for protein identification by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry at Tianjin Biochip Corporation (Tianjin, China).…”

“…Escherichia coli and Pichia pastoris ) [ 8 , 16 ] has some disadvantages like high equipment cost, high energy consumption, serious environment pollution, complex processing, etc. An efficient and economic alternative is to produce α-galactosidase directly in feed grains as β-mannanase and xylanase produced in transgenic maize [ 17 ] and rice [ 18 ], respectively.…”

Plant Molecular Farming: Much More than Medicines

“…MEY-1 β-mannanase under the control of an embryo-specific promoter were tested because Bispora is an acidophile and the enzyme should therefore remain functional in the low-pH gastric environment. The enzyme was found to remain active under the same conditions as the commercial supplement from P. pastoris but was more thermotolerant during pelleting, with an activity of 10,000 U/kg (87). A feeding trial of β-mannanase supplements in pigs revealed that 400 U/kg feed achieved the greatest feed efficiency, so 40 g of the transgenic seeds per kilogram of conventional diet is sufficient to achieve the anticipated effects (88).…”

“…The enzyme was also resistant to gastric digestion allowing it to act on phytate in the gut. Following successful field trials in chickens, phytaseenhanced maize was released as China's first transgenic crop in 2009, and the biosafety certificate was renewed in 2015 (87).…”