Studies for the Genetic Engineering in Sweetpotato (Ipomoea Batatas L.) for Starch Bioconversion

Abstract

SANTA-MARIA, MONICA CECILIA. Studies for the Genetic Engineering of Sweetpotato for Starch Bioconversion. (Under the direction of Bryon Sosinski and William F. Thompson).

Concerns over energy security and reducing greenhouse gas emissions have triggered interest in alternative, carbon-neutral, renewable energy sources. One alternative is biofuels for use in transportation. In the U.S., ethanol dominates the biofuel market and is mostly obtained from cornstarch. Due to sustainability concerns over extensive corn utilization, identification of alternative biofuel feedstocks is necessary. The high starch content in storage roots along with agronomical advantages make sweetpotato (Ipomoea batatas L.) and attractive biofuel feedstock in the southeast U.S. Industrial starch conversion to fermentable sugars involves a liquefaction step where starch is solubilized and partially hydrolyzed at high temperatures by a thermostable and thermoactive α-amylase. Monomeric sugars are obtained during saccharification by further enzymatic activity. These enzymes are added to the starch mixture increasing overall process economics. To make starch processing more cost-effective, hyperthermophilic α-amylase from Thermotoga maritima was introduced into the sweetpotato genome to allow for starch self-processing at high temperatures. Hyperthermophilic α-amylase production was fist tested in tobacco cell cultures as model plant system. Functional enzyme was produced exhibiting enhanced thermostability, but otherwise identical biochemical properties compared to recombinant production in Escherichia coli. The enhanced stability of plant-made enzyme was due to intrinsically provided calcium in plant cells. This opened prospects of further cost reduction by eliminating need for calcium addition. The Tma α-amylase gene was then introduced into the sweetpotato genome by stable transformation with Agrobacterium tumefaciens. Starch in transgenic storage roots was readily hydrolyzed at 80°C, while starch in wild type roots remained unchanged. No recombinant enzyme activity was detected at ambient temperatures and transgenic storage roots developed normally. The feasibility of biomass conversion was thus demonstrated. Additional work was undertaken to expand transformation technology to novel high starch sweetpotato varieties for increased bioethanol yields. Optimized regimes for in vitro regeneration in selected genotypes were established. Alternatively, storage root specific promoters were identified from a sweetpotato genomic library to be used for targeted expression of recombinant enzymes to sweetpotato storage roots.