The development of a cost-competitive bioprocess requires that the cell factory converts the feedstock into the product of interest at high rates and yields. However, microbial cell factories are exposed to a variety of different stresses during the fermentation process. These stresses can be derived from feedstocks, metabolism, or industrial production processes, limiting production capacity and diminishing competitiveness. Improving stress tolerance and robustness allows for more efficient production and ultimately makes a process more economically viable. This review summarises general trends and updates the most recent developments in technologies to improve the stress tolerance of microorganisms. We first look at evolutionary, systems biology and computational methods as examples of non-rational approaches. Then we review the (semi-)rational approaches of membrane and transcription factor engineering for improving tolerance phenotypes. We further discuss challenges and perspectives associated with these different approaches.

The flavonoid pinocembrin and its derivatives have gained increasing interest for their benefits on human health. While pinocembrin and its derivatives can be produced in engineered Saccharomyces cerevisiae , yields remain low. Here, we describe novel strategies for improved de novo biosynthesis of pinocembrin from glucose based on overcoming existing limitations in S. cerevisiae. First, we identified cinnamic acid as an inhibitor of pinocembrin synthesis. Second, by screening for more efficient enzymes and optimizing the expression of downstream genes, we reduced cinnamic acid accumulation. Third, we addressed other limiting factors by boosting the availability of the precursor malonyl-CoA, while eliminating the undesired byproduct 2′,4′,6′-trihydroxy dihydrochalcone. After optimizing cultivation conditions, 80 mg/L pinocembrin was obtained in a shake flask, the highest yield reported for S. cerevisiae . Finally, we demonstrated that pinocembrin-producing strains could be further engineered to generate 25 mg/L chrysin, another interesting flavone. The strains generated in this study will facilitate the production of flavonoids through the pinocembrin biosynthetic pathway.

Background Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. Conclusion This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

Background Two important flavonoids, kaempferol and quercetin possess remarkably potent biological impacts on human health. However, their structural complexity and low abundance in nature make both bulk chemical synthesis and extraction from native plants difficult. Therefore microbial production via heterologous expression of plant enzymes can be a safe and sustainable route for their production. Despite several attempts reported in microbial hosts, the production levels of kaempferol and quercetin still stay far behind compared to many other microbial-produced flavonoids. Results In this study, Saccharomyces cerevisiae was engineered for high production of kaempferol and quercetin in minimal media from glucose. First, the kaempferol biosynthetic pathway was reconstructed via screening various F3H and FLS enzymes. In addition, we demonstrated that amplification of the rate-limiting enzyme AtFLS could reduce the dihydrokaempferol accumulation and improve kaempferol production. Increasing the availability of precursor malonyl-CoA further improved the production of kaempferol and quercetin. Furthermore, the highest amount of 956 mg L− 1 of kaempferol and 930 mg L− 1 of quercetin in yeast was reached in fed-batch fermentations. Conclusions De novo biosynthesis of kaempferol and quercetin in yeast was improved through increasing the upstream naringenin biosynthesis and debugging the flux-limiting enzymes together with fed-batch fermentations, up to gram per liter level. Our work provides a promising platform for sustainable and scalable production of kaempferol, quercetin and compounds derived thereof.

BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to address if improvements in terms of yield, ease of production and if purification advantages can be identified. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, showed to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind human HER3 as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching high yields and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.