Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization

Abstract

Background: Mannans 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 findings: The 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/significance: This 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.

Figure 1. Construction of the recombinant vector and regeneration of transgenic maize.

A) The recombinant expression vector pHP20754-man5As. B) The chimeric gene cassettes for expression in maize. C) PCR analysis of five putative calli. Lane 1, the DNA molecular weight markers; lane 2, the expression vector pHP20754-man5As (positive control); lane 3–7, the calli of transgenic maize Hi-II; lane 8, the calli of non-transgenic maize Hi-II (negative control). D) Embryogenic calli in selective medium. E) Plantlets in rooting medium. F) Regenerated maize plants in the greenhouse. G) Transgenic maize in fields. H) Ears of generation T1 of transgenic plant and non-transgenic maize Zheng58. I) Seeds of generation T1 of transgenic plant and non-transgenic maize Zheng58.

Figure 2. PCR analysis of the genomic DNA from leaves of generation BC1 of transgenic and non-transgenic plants.

A) PCR detection of the gene man5As. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the vector pHP20754-man5As; lane 12, the non-transgenic Zheng58. B) PCR detection of the gene actin. Lane 1, the DNA molecular weight markers; lane 2–10, the transgenic plants; lane 11, the non-transgenic Zheng58.

Figure 3. Southern blot analysis of MAN5AS in three transgenic plants of event 22 after digestion with HindIII and BamHI.

Lane 1, the DIG-labeled molecular weight markers; lane 2–4, MAN5AS with BamHI digestion; lane 5 and 6, non-transgenic Zheng58 digested by BamHI and HindIII, respectively; lane 7–9, MAN5AS with HindIII digestion; lane 10, the digested expression cassettes as a positive control.

Figure 4. Analysis of recombinant MAN5AS from two transgenic maizes.

A) SDS–PAGE. B) Western blot. Lane 1 and 3, the transgenic maize; lane 2, the protein molecular markers; lane 4, the non-transgenic Zheng58; lane 5, the purified MAN5A-SST produced in P. pastoris. C) Specific promoter analysis. Lane 1, the protein molecular markers; lane 2 and 4, the protein isolated from seeds of T042-5 and T041-20; lane 3 and 5, the two transgenic plant proteins pretreated with Endo H; lane 6, the protein isolated from seeds of non-transgenic Zheng58; lane 7, the purified MAN5A-SST produced in P. pastoris; lane 8–10, the proteins collected from root, stem and leaf tissue of a transgenic plant.

Figure 5. Property comparison of recombinant β-mananases expressed in maize (MAN5AS) and P. pastoris (MAN5A-SST).

a) pH-dependent activity profiles of MAN5A-SST and MAN5AS at 65°C. b) pH stability of MAN5A-SST and MAN5AS activities at 37°C for 1 h. c) Temperature-dependent activity profiles of MAN5A-SST and MAN5AS at pH 1.5. d) Thermostability of MAN5A-SST (dashed line) and MAN5AS (solid line) at 60°C (diamond) or 90°C (square) at pH 1.5. Error bars represent the standard deviation of triplicate measurements.