Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol.

Aleksandra Gęsicka,Monika Borkowska,Wojciech Białas,Ewelina Celińska,Paulina Kaczmarek

doi:10.3390/microorganisms8050717

Aleksandra Gęsicka, Monika Borkowska + Show 3 more

Open Access

https://doi.org/10.3390/microorganisms8050717

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Journal: Microorganisms	Publication Date: May 12, 2020
Citations: 7	License type: CC BY 4.0

Affiliation: University of Life Sciences in Poznań

Abstract

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

Highlights

Starch is one of the most abundant carbohydrates, second only to cellulose, found in higher plant biomass
Production of the target polypeptides was accompanied by high biomass growth (59.03 ± 7.67 g/L) and synthesis of small molecular weight metabolites typical for Y. lipolytica cultures under adopted conditions
We used Y. lipolytica as a heterologous protein expression platform to produce the RSD-amylolytic preparation composed of two enzymatic activities: alpha-amylase SoAMY and glucoamylase TlGAMY

Summary

Introduction

Starch is one of the most abundant carbohydrates, second only to cellulose, found in higher plant biomass. Valorization of starch-containing waste streams produced in massive amounts from confectionery manufacturing and bakeries, as well as discarded, damaged, or out of date products that return on site, were recently indicated as a largely ignored trend, with a huge potential for bioprocessing [2,4] Up to date, these waste streams have been valorized through composting, as animal feed or disposed in landfills, and could be used as sustainable resource in microbial production of high-value added products. The starch-containing feedstock is gelatinized by thermal treatment and liquefied by thermostable α-amylase at 105 ◦C, and after cooling down to 60 ◦C, liquefied starch is saccharified to fermentable sugars by glucoamylase [5], which can be further converted to desired biomolecules This initial thermal treatment generates high energy consumption, largely increasing the overall process cost and market price of the final product [6,7,8]. Novel biocatalysts capable of digesting raw, non-pre-treated starch at lower temperatures are being sought [10]

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