Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2×YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2×YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Investigation of Enzymatic Hydrolysis Kinetics of Soy Protein Isolate: Laboratory And Semi-Industrial Scale

Soy protein isolate is a worthy substitute for meat protein. However, its low level of digestibility limits its spread to new market niches. This problem can be solved by enzymatic hydrolysis of soy protein to peptides. Several research teams have already been solving this problem, but their results were obtained under laboratory conditions and do not provide information about the reproducibility of the results on an industrial scale. In this paper, we have compared the results of laboratory and semi-industrial experiments of enzymatic hydrolysis of protein. Also the kinetics of the reaction under different conditions is shown, and the final product is characterized. The obtained results of semi-industrial experiments can form the basis of industrial regulations for the production of soy protein hydrolysate as an easily digestible form of dietary protein for athletes and patients with digestive disorders.

Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

Following the Thread: Elite Iconography on Weaving Objects at Poggio Civitate (Murlo)

Evidence for industrial scale production of numerous manufacturing processes has been attested in all phases of occupation at Poggio Civitate (Murlo). A subset of these, tools for the production of textiles and fibers, indicates that textile crafts were manufactured on a large scale as a part of a centralized and organized industry. These industrialized practices occurred within and around the monumental seventh and sixth century BCE complexes which displayed architectural decoration bearing iconographic themes that served to secure the positions of the aristocratic elites. This paper investigates the stamped decoration present on rocchetti and its relationship to the architectural decoration present on the monumental structures of the site. As small moveable objects used by members of the community, rocchetti present an opportunity to investigate the movement of elite images through the non-elite population of a community and their reception of aristocratic ideology presented in monumental structures.

Long-Term Assessment of Temperature Management in an Industrial Scale Biogas Plant

Temperature management is one of the primary considerations of biogas plant operation, and influences physical and biochemical processes. An increase in the temperature leads to an increase in the hydrolysis rate of the feedstock, while it can inhibit microorganisms taking part in different stages of anaerobic digestion. Because of the complexity of the biochemical processes within the anaerobic digestion process, there is a lack of knowledge about the effects of temperature and temperature change on efficiency. Moreover, the impact of stirring directly affects the temperature distribution in the anaerobic digestion reactors. In this study, the temperature management in an industrial-scale biogas plant was examined, and the effect of small temperature changes (from the operation temperature 42 °C) on the efficiency was studied in a laboratory under two different conditions: with stirring (at 40 and 44 °C) and without stirring (at 40 and 44 °C). The examination results from the biogas plant showed that heat transfer in the reactor was not sufficient at the bottom of the digester. Adaptation of the post-digester samples to the temperature changes was more challenging than that of the digester samples. From digestate samples, higher biomethane generation could be obtained, resulting from sufficient contact between microorganisms, enzymes, and substrates. Overall, differences between these changing conditions (approx. 6 NmL CH4 g VS−1) were not significant and could be adapted by the process.