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Development of a xylose-utilizing Saccharomyces cerevisiae cell factory for the production of muconic acid and protocatechuic acid

Book - Dissertation

Today, society is shaped by fossil fuels and its widespread utilization, which impacts the Earth's ecosystems and leads to the anthropogenic climate change. Many products that we use on a daily basis are made of chemicals derived from fossil fuels. The shift from a fossil-resource based economy to a more sustainable, bio-based economy requires the development of alternative production routes that utilize cheap renewable biomasses, such as lignocellulose. The yeast Saccharomyces cerevisiae has extensively been engineered to efficiently convert both hexose and pentose sugars in lignocellulosic hydrolysates towards ethanol. These strains are also ideal candidates to construct cell factories for other platform chemicals. There are many interesting platform chemicals but in this work we focus on two: muconic acid and protocatechuic acid (PCA). Muconic acid can be converted into adipic and terephthalic acid, which are two acids currently produced on an industrial scale but rely on fossil-based resources. Moreover, muconic acid's two double bonds offer great versatility for chemical modification. PCA can serve as a commodity for the production of catechol or benzene. The use of cell factories to produce non-native chemicals has been investigated extensively, and also specifically for muconic acid. However, the production titers and yields needed for industrial implementation are far from being achieved and the best-performing bacterial cell factories are not preferred due to their inferior robustness in industrial settings. This is why second-generation industrial S. cerevisiae strains are considered more promising alternatives. In the first part of this work, we constructed a xylose-utilizing yeast cell factory to produce muconic acid. We successfully integrated and expressed six copies of the muconic acid pathway (MApw), consisting of three heterologous genes, and abolished ethanol production by deletion of the three PDC genes in the industrial yeast strain T18HAA1. Furthermore, we improved the conversion of the heterologous intermediate PCA to muconic acid by increasing the co-factor production and increasing the PCA decarboxylase (PCAD) expression, which resulted in the best performing strain TN22. This strain produced maximal muconic acid titers of 4,000 mg/L (33.6 mg/g carbon), 4,500 mg/L (37.3 mg/g carbon) and 3,800 mg/L (30.7 mg/g carbon) in medium containing glucose, xylose or a mixture of the two, respectively. To date, these are the highest reported titers of muconic acid production in batch fermentations using yeast as a cell factory. However, we found that muconic acid has an inhibitory effect on sugar consumption, growth and muconic acid production, especially at lower pH levels. In-situ product removal with polypropylene glycol (PPG) lowered the muconic acid concentration in the fermentation medium, but it was not enough to recover the fermentation capacity. Further observations showed that the cause of fermentation arrest is not only due to the toxicity of muconic acid but also an unknown, possible metabolic bottleneck. With the TN22 strain, we also achieved a titer of 2,900 mg/L (26.4 mg/g carbon) muconic acid from corn cob lignocellulose hydrolysate with addition of PPG. In the second part of this work, we used similar strategies for the construction of a xylose-utilizing yeast cell factory to produce PCA. Additionally, the carbon flux towards PCA was improved and resulted in the TN23 ric1Δ/RIC1 strain. This strain produced maximal titers of 7,700 mg/L (65 mg/g carbon), 5,200 mg/L (43 mg/g carbon) and 6,700 mg/L (54 mg/g carbon) in medium containing glucose, xylose or a mixture of the two, respectively. Incomplete sugar utilization and fermentation arrest was also observed. This is the first report of a yeast cell factory producing PCA as the target chemical. In both cases, the abolishment of ethanol production in the developed strains necessitates medium supplementation with a C2 compound such as ethanol. The targeted engineering strategies used to develop a C2 independent strain did not improve the strain's performance, which prompts further investigation. Both the TN22 and TN23 ric1Δ/RIC1 strains can serve as preliminary platform strains for muconic acid or PCA production, respectively, with further investigation of end-product toxicity, C2 auxotrophy and improved sugar utilization capacity.
Publication year:2022
Accessibility:Embargoed