Metabolic engineering of Saccharomyces cerevisiae for efficient ethanol production from pentose sugars

Biofuels;renewable energy;lignocellulosic ethanol;pentose utilization;combinatorial engineering;Deoxyribonucleic acid (DNA) assembler;metabolic engineering;synthetic biology;system biology

Du, Jing

关键词: Biofuels;    renewable energy;    lignocellulosic ethanol;    pentose utilization;    combinatorial engineering;    Deoxyribonucleic acid (DNA) assembler;    metabolic engineering;    synthetic biology;    system biology;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/45695/Du_Jing.pdf?sequence=4&isAllowed=y

美国|英语

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

【 摘 要 】

In recent years, an increasing desire to develop a feasible alternative to fossil fuels has led to an increased amount of attention in the area of bioconversion—particularly that of converting plant-derived lignocellulosic material into biofuels. Saccharomyces cerevisiae, also known as baker’s yeast, is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which constitute up to 30% of biomass hydrolysate, cannot be utilized by S. cerevisiae. To this end, heterologous pentose utilization pathways have been introduced into S. cerevisiae in order to enable the assimilation of pentose sugars. However, pentose utilization of recombinant S. cerevisiae strains are inefficient due to the poor sugar uptake ability, the low expression level and activity of heterologous proteins, the redox imbalance that results from different cofactor preference in oxidation and reduction reactions, and a suboptimal metabolic flux through different catalytic steps. A lot of research has been carried out to improve pentose utilization in S. cerevisiae by targeting different aspects of these issues, but it is very challenging to come up with a single strategy which can solve all three problems at the same time. To address problem of inefficient pentose sugar uptake in S. cerevisiae, a collection of eighteen putative pentose transporters were cloned. Among them, one arabinose-specific and two xylose-specific transporters from Pichia stipitis and Neurospora crassa were identified. All transporters were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and immunofluorescence microscopy, respectively. Overexpression of the xylose-specific transporters under weak promoters was shown to help xylose fermentation in xylose assimilating S. cerevisiae. To optimize the heterologous xylose utilization pathway, both an enzyme-based strategy and a promoter-based strategy were applied to balance the metabolic flux for xylose utilization. For the enzyme-based strategy, around twenty different enzyme homologues from various fungal species with different catalytic efficiency and cofactor preference were cloned for each catalytic step. These enzyme homologues were assembled into a library of xylose utilization pathways using our newly developed DNA assembler method. Using the library we generated, clones with better combinations of enzyme homologues were identified using a colony size based screening method.To fine-tune the expression level of the three genes in a heterologous xylose utilization pathway, enzyme homologues of xylose reductase, xylitol dehydrogenase and xylulokinase with the highest activity and matched cofactor specificity were selected and promoter mutants with varying strengths were randomly incorporated into the pathway using DNA assembler. Using this method, a number of xylose utilization pathways with optimized expression levels were identified using a high throughput screening method. Additionally, this method was also applied to directly search for an optimized xylose utilizing pathway for industrial yeast strains with different metabolic flux patterns compared to laboratory S. cerevisiae strains. Optimized xylose utilizing pathway mutants were also identified in industrial strains and their gene expression levelswere actually different from that of the laboratory strain.To facilitate the metabolic engineering in industrial strains with the new pathway assembly strategy, multiple expression and integration vectors were constructed. These vectors require no modification on the industrial host strains by using positive selection markers. Moreover, by keeping the homologous region used for pathway assembly in the laboratory vectors, the gene expression cassettes for pathway optimization can be directly used in the industrial strains. In addition to the gene expression systems, a scar-less gene modification method was also investigated in the industrial strains and promising preliminary results were obtained.By introducing a new pentose-specific transporter, optimization of the heterologous pentose utilization pathway and the utilization of robust industrial polyploid host strains, the efficiency of pentose sugars in recombinant S. cerevisiae strains were significantly improved. More efficient utilization of pentose sugars will lead to more efficient utilization of biomass feedstocks and therefore lower the cost for lignocellulosic ethanol production.