Engineering and evaluation of yeast strains for the production of lactic acid from cellulosic sugars

Yeast;Lactic acid;Metabolic engineering;JEN1;ADY2;Xylose;Cellobiose;Lignocellulosic;Saccharomyces cerevisiae

Turner, Timothy Lee

关键词: Yeast;    Lactic acid;    Metabolic engineering;    JEN1;    ADY2;    Xylose;    Cellobiose;    Lignocellulosic;    Saccharomyces cerevisiae;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/92933/TURNER-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Terrestrial biomass consists largely of lignocellulosic materials. Abundant in nature, lignocellulosic biomass can be cultivated easily on land otherwise unsuitable for traditional crops or be harvested from crop waste, such as corn stover. After pretreatment and hydrolysis, the lignocellulosic biomass will yield several sugars including glucose (6C), xylose (5C) and cellobiose (a glucose dimer). Complete conversion of each of these sugars is necessary to efficiently produce a target fermentation product from lignocellulosic hydrolysates. Until now, biofuels, such as ethanol, have been the only major fermentation product produced at an industrial-scale from lignocellulosic biomass. The primary goal of my dissertation research is to utilize metabolic engineering to construct a recombinant microbe capable of rapidly and efficiently producing value-added non-ethanol products from these lignocellulosic sugars. With this in mind, lactic acid has been selected as the target product to develop this lignocellulosic chemical production platform. Lactic acid has many industrial uses including as a feedstock for surgical implants, 3D printing, and renewable polyesters. The production of lactic acid from xylose was first achieved by cloning and introduction of a heterologous lactate dehydrogenase gene (ldhA) from the filamentous fungus Rhizopus oryzae into an engineered, xylose-fermenting Saccharomyces cerevisiae yeast strain. Simultaneous co-fermentation of xylose and cellobiose for the production of lactic acid by yeast was achieved by the introduction of the ldhA cassette into an engineered, xylose- and cellobiose-fermenting S. cerevisiae. Through screening on a variety of fermentation conditions and carbon sources, it was determined that non-repressing sugars, such as xylose and cellobiose, resulted in high lactic acid yields and negligible ethanol yields despite no genotypic disruption of the native yeast ethanol pathways. Similarly, a high lactic acid yield was also achieved from lactose and cheese whey by the ldhA-expressing yeast strain. Production from lignocellulosic feedstocks was scaled-up to a one-liter bioreactor as a proof-of-concept, resulting in lactic acid yields exceeding 70 g lactic acid/g sugar with titers greater than 120 g/L of lactic acid. To further improve lactic acid yield by the engineered strains, a variety of industrial S. cerevisiae yeast strains were screened for their ability to tolerate organic acids, low pH conditions, and common fermentation inhibitors such as furfural. The industrial strain screening provided insight towards the future development of a highly lactic acid-resistant Saccharomyces spp. strain. In addition to attempting to improve lactic acid production by selecting an ideal parental yeast strain, several metabolic engineering approaches were implemented to elucidate ideal genetic characteristics of an engineered ldhA-expressing strain. Primarily, it was discovered that deletion of JEN1 and/or ADY2, genes which code for monocarboxylate transporters, either reduced lactic acid uptake (∆ADY2ΔJEN1 or ΔJEN1) or reduced lactic acid yield by at least 25 % (∆ADY2ΔJEN1). Collectively, this research has demonstrated a viable platform for the production of non-fuel chemicals from lignocellulosic feedstocks by engineered yeast and generated new understanding for the molecular basis of lactic acid production by engineered microbes.