Soluble and insoluble & alpha;-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm

Molecular mechanisms that distinguish the synthesis of semi-crystalline & alpha;-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched & alpha;-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate & alpha;-glucan accumulation. PHO did not support & alpha;-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline & alpha;-glucan superstructures up to 4 & mu;m in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence. Expressing up to 11 maize starch biosynthesis enzymes in yeast results in the production of insoluble and soluble glucose polymers that resemble plant starch molecules in many respects.

Automated summary

By using yeast as a test bed, the researchers isolated starch biosynthetic enzymes from maize endosperm and found that different combinations of enzymes led to variations in the accumulation of soluble and insoluble starch, shedding light on the molecular mechanisms that differentiate plant starch from nonplant species.