An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

Repeated glucose oscillations in high cell-density cultures influence stress-related functions of Escherichia coli.

Engineering microbial cells for the commercial production of biomolecules and biochemicals requires understanding how cells respond to dynamically changing substrate (feast-famine) conditions in industrial-scale bioreactors. Scale-down methods that oscillate substrate are commonly applied to predict the industrial-scale behavior of microbes. We followed a compartment modeling approach to design a scale-down method based on the simulation of an industrial-scale bioreactor. This study uses high cell-density scale-down experiments to investigate Escherichia coli knockout strains of five major glucose-sensitive transcription factors (Cra, Crp, FliA, PrpR, and RpoS) to study their regulatory role during glucose oscillations. RNA-sequencing analysis revealed that the glucose oscillations caused the down-regulation of several stress-related functions in E. coli. An in-depth analysis of strain physiology and transcriptome revealed a distinct phenotype of the strains tested under glucose oscillations. Specifically, the knockout strains of Cra, Crp, and RpoS resulted in a more sensitive transcriptional response than the control strain, while the knockouts of FliA and PrpR responded less severely. These findings imply that the regulation orchestrated by Cra, Crp, and RpoS may be essential for robust E. coli production strains. In contrast, the regulation by FliA and PrpR may be undesirable for temporal oscillations in glucose availability.

Utilization of plant derived lactic acid bacteria for efficient bioconversion of brewers' spent grain into acetoin.

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive.

Development of a yoghurt alternative, based on plant-adapted lactic acid bacteria, soy drink and the liquid fraction of brewers' spent grain.

With consumers becoming more aware of sustainability, healthier lifestyles and animal welfare, plant-based food products as alternatives to dairy products have become a fast-growing industry in the last decade, and an increasing number of plant-based products are showing up on the markets. With over 88 million tons of food wasted in Europe annually, a sustainable alternative to dairy could be to use side streams for new products. Here, we tried to develop a plant-based yogurt alternative based on three ingredients: commercial soy drink and a liquid fraction of brewers' spent grain fermented with plant-adapted lactic acid bacteria. Analysis of the content and properties of the fermented product were compared to a commercial plant-based yoghurt-like product and a commercial dairy yoghurt. Results from the project show that fermentation of a commercial soy drink containing 20% of the liquid fraction of brewers' spent grain results in a product with texture and sensory characteristics that mimics a dairy yogurt.

Improvement of cis,cis-Muconic Acid Production in Saccharomyces cerevisiae through Biosensor-Aided Genome Engineering.

Muconic acid is a potential platform chemical for the production of nylon, polyurethanes, and terephthalic acid. It is also an attractive functional copolymer in plastics due to its two double bonds. At this time, no economically viable process for the production of muconic acid exists. To harness novel genetic targets for improved production of cis,cis-muconic acid (CCM) in the yeast Saccharomyces cerevisiae, we employed a CCM-biosensor coupled to GFP expression with a broad dynamic response to screen UV-mutagenesis libraries of CCM-producing yeast. Via fluorescence activated cell sorting we identified a clone Mut131 with a 49.7% higher CCM titer and 164% higher titer of biosynthetic intermediate-protocatechuic acid (PCA). Genome resequencing of the Mut131 and reverse engineering identified seven causal missense mutations of the native genes (PWP2, EST2, ATG1, DIT1, CDC15, CTS2, and MNE1) and a duplication of two CCM biosynthetic genes, encoding dehydroshikimate dehydratase and catechol 1,2-dioxygenase, which were not recognized as flux controlling before. The Mut131 strain was further rationally engineered by overexpression of the genes encoding for PCA decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1pΔE), and by restoring URA3 prototrophy. The resulting engineered strain produced 20.8 g/L CCM in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, representing the highest reported performance metrics in a yeast for de novo CCM production to date and the highest production of an aromatic compound in yeast. The study illustrates the benefit of biosensor-based selection and brings closer the prospect of biobased muconic acid.