During traditional alcohol fermentation process, exogenous enzymes are required to be added (Dutta et al.), however, the cost of exogenous enzymes is much too high. It was found that the cost contribution of exogenous enzymes to ethanol produced during the conversion of corn starch was $0.68/gal under the condition that the sugars in the biomass could be converted at maximum theoretical yields. Furthermore, that would be $1.47/gal if the yields were based on saccharification and fermentation yields, leading to conclusion that considerably efforts are in need compulsorily to reduce the cost of exogenous enzymes (Klein-Marcuschamer et al.).
Sweet potato residue (SPR) is a byproduct generated after sweet potatoes are converted into starch. Approximately 4.5–5.0 tons of fresh SPRare generated for 1 ton of starch produced (Zhu et al.). However, most of SPRs are directly discarded though a small amount of them is used to feed animals, resulting in heavy environmental burden(Liu et al.; Jin et al.). It is reported that SPR is composed of starch (51.98%), dietary fiber (21.41%) and protein (4.55%) (dry basis) (Mei, Mu and Han). It can serve as renewable lignocellulosic raw material (a complex mixture of carbohydrates) that requires accessible pathway to enzymes for producing fermentable sugars which can be fermented into ethanol after hydrolysis, leading to sustainability (Busic et al.). Furthermore, some studies do concentrate on converting the SPRs into economical resources (Dong, Mu and Sun), such as resources for animal feed production (Serena, Jorgensen and Knudsen; Yde et al.), biomass production (SINGH; Klingspohn et al.; Hashem and Darwish; Yokoi et al.), and component extraction (NODA et al.; Yoshimoto, Yamakawa and Tanoue; Mei, Mu and Han; Zhang and Mu). However, these usages are confronted with some limits such as low-efficiency, high cost, and incomplete utilization of SPRs. To introduce the optimal usage of SPRs, some researches indicated that SPRs can be converted into high yields of microbial protein within short fermentation time (Fields, Tantratian and Baldwin; Ghanem; Manilal, Narayanan and Balagopalan; Pasari, Korus and Heimsch), proving that SPRs can be an excellent substrate for microorganisms to proceed fermentation. Therefore, microbial fermentation of SPRs should be the optimal method to valorize wasted SPRs, and this fact is also one of the inspirations of our team project.
To specify our inspiration further, we did overall research on enzymatic hydrolysis method for ethanol fermentation, and it is reported that the condition of enzymatic hydrolysis of SPRs is milder, more efficient and suitable to industrial application, its experiments also indicate that the best method to hydrolyze SPRs is the multienzyme function (徐帅 et al.). What is more, it was reported that integration of glucoamylase genes can reduce at least 40% of the dose of glucoamylase (Wang et al.), indicating that integration of genes of two enzymes might have greater potential and effect of reducing (over 40%) exogenous enzymes according to the multienzyme function experiment (徐帅 et al.), and amylase does play a crucial role in starch fermentation (Cripwell et al.; Favaro et al.).
Therefore, we identified our two target enzymes; amylase and glucoamylase combination which were proved to work efficiently (Sakwa et al.; Eksteen et al.; Kim et al.; Yamakawa et al.; de Moraes, Astolfi-Filho and Oliver). Moreover, our project chosen is inspired by the team of 2021 iGEM (Fujian_United) research on yeast secreting glucoamylase and the whole conception is finalized with the help of Dr. WANG XIN ( Lecture, Henan University of Technology,) and our instructor Ms. Bell Duan (SubCat Academy).
To recall, the problems we are going to solve are that the cost of exogenous enzymes during alcohol fermentation process is much too high, and wasted sweet potato residue is harmful to the environment. Therefore, as long as we enable the saccharomyces cerevisiae to self-secrete alpha-amylase and glucoamylase which function to completely hydrolyze starch into glucose molecules through synthetic biology, then we can largely reduce the cost of exogenous enzymes, and we can also put sweet potato residue into use as a raw material of alcoholic fermentation to turn the pollution problems into profits and efficiency smartly. To be more specific, we will transform plasmids containing genes (GA, temA) that express alpha-amylase and glucoamylase into our yeast 1974.
how does our project work to turn starch into glucose molecules to reduce cost of exogenous enzymes (made in Canva.cn)
Overview of the methodology of our project design through synthetic biology (made in Canva.cn)
Based on the conception of synthetic biology, our group aims at successfully constructing saccharomyces cerevisiae strain which is able to self-secrete amylase and glucoamylase through the methodology of molecular biology. Alpha-amylase and glucoamylase protein expressed by GA and temA with biological functions could be obtained from 1974. SDS-PAGE, enzymatic activity assay and et al show that the protein has good activity. Furthermore, the engineered strain will be applied to help ferment sweat potato residue for alcoholic production so that we can effectively achieve utilizing and transforming sweat potato residue which is previously wasted into profitable value. In the meantime, we will also help reduce the cost of exogenous enzymes in the alcoholic fermentation process.
Our goal diagram (made in Canva.cn)
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