Exploring Saccharomyces cerevisiae in the light of novel small-scale cultivation techniques

The fast evolving field of industrial biotechnology focuses on the development of bioprocesses and products valuable for society and economy. An integral part of a given bioprocess are microorganisms that are turned into efficient microbial cell factories to synthesize a desired product at high titers, yields and rates. Advances in synthetic biology tools focus on the development of microbial strain design and its purposeful construction, presenting microbial cell factories with novel artificial biological pathways or improved features for efficient product formation. Such modifications frequently intervene in the natural flux of metabolites as well as in the energy and redox metabolism of the microorganism. This leads to significantly different cultivation parameters, in terms of substrate uptake, growth and (by-)product formation kinetics, than experienced for the respective wild-type strain. In consequence, the physiology of engineered strains may vary considerably from cell factory to cell factory.
These evident differences in strain physiology paired with the increasing efficiency of synthetic biology tools to generate large strain libraries of producer variants create the need for quantitative high-throughput cultivation systems for i) screening approaches to select the most promising production strain and ii) cultivation techniques to investigate process variables and conditions beneficial for the synthesis of a desired product.
The first study demonstrates the establishment of a fed-batch mimicking medium tailored for the robust cultivation of Saccharomyces cerevisiae in a small-scale system based on microtiter plates. This system was designed for simple high-throughput screening applications, allowing the acquisition of data sets that are comparable to values obtained in bench-scale stirred tank reactors. Here, the key parameters comprised a carbon-limited cultivation regime for controlling the growth rate and pH, as both are of importance in common cultivation strategies for the reproducible cultivation of microorganisms. A carbon-limited cultivation regime was achieved by applying a polysaccharide gradually releasing glucose monomers upon enzymatic cleavage, enabling fed-batch mode through substrate-limiting conditions. The pH was, unlike in bioreactors, not maintained through acid and base addition, but with a suitable buffer agent. Respective buffer agents were chosen for a near-neutral pH of 6.4 and a low pH of 3.8. Iterative steps were taken towards the development of a small-scale cultivation system operating in fed-batch mode for S. cerevisiae cultivation, including the choice of a suitable substrate, buffer and enzyme concentration, as well as appropriate inoculum size. Two engineered S. cerevisiae strains, synthesizing the industrially relevant product 3-hydroxypropionic acid (3-HP), were used to validate this system by comparing obtained cultivation parameters with values determined in stirred tank bioreactors. Similar product yields on glucose were revealed, such as e.g. 0.052 ± 0.002 g·g-1 in microtiter plates for S. cerevisiae strain ST687 at pH 6.4 and 0.053 ± 0.001 g·g-1 for the respective bioreactor cultivation. In spite general insecurities about the predictability and reliability of results obtained from stirred tank reactors based on cultivations in microtiter plates, yields as sensitive parameter for strain selection were found to be comparable in both microtiter plates and bioreactors. The small-scale fed-batch mimicking system described in this study is a simple tool for various screening applications at near-neutral and low pH, enabling high-throughput due to its easy use in microtiter plates. Furthermore, in contrast to commercially available systems, all details for the strategy to enable microbial cultivation in fed-batch mode are disclosed in this study, which allows its adaptation for the use of other microbial cell factories that utilize glucose as carbon source.
The second study demonstrates the use of a parallelized small-scale chemostat cultivation system for the quantitative physiological characterization of a S. cerevisiae strain engineered for 3-HP production. As chemostats are an ideal tool for systematic physiological investigation of microorganisms enabling steady state conditions due to the precise control of the growth rate, steady substrate and (by-)product concentrations can be measured as well as corresponding yields and rates determined. This otherwise very time consuming and laborious approach was addressed by the high degree of parallelization of the small-scale chemostat system, of up to 24 cultivation vessels, applied in this study. Thus, a parallelized small-scale chemostat system was used to investigate different growth rates and substrate limitations (carbon, phosphate, nitrogen) with respect to product formation. Applying carbon-limitation, the highest 3-HP yield on glucose was achieved at the lowest growth rate tested, as well as the highest yields of by-products formed. Furthermore, the effect of phosphate- or nitrogen-limitation on product formation was investigated, since it was shown in previous studies that secondary substrate limitations are able to force the microorganism into a beneficial state, promoting the synthesis of the desired product. Phosphate-limitation emerged to be beneficial for 3-HP synthesis achieving a product yield on glucose of 0.211± 0.018 g·g-1, which is approx.
19 % higher than the value obtain from carbon-limiting conditions. These findings were used to design a fed-batch process, which is commonly preferred in industrial setting due to the achievement of high product yields per volume with this cultivation strategy. Therefore, fed-batch cultivations were performed under conditions comparable to the chemostat cultivations. Product yields on glucose of 0.211± 0.018 g·g-1 determined in chemostats at a growth rate of 0.04 h-1 applying phosphate-limitation and 0.256 ± 0.017 g·g-1 obtained in stirred tank bioreactors in fed-batch mode at a marginally higher growth rate of 0.05 h-1 show comparable values. Similar results were calculated for carbon-limitation, revealing a yield of 0.166 ± 0.017 g·g-1 in chemostats and 0.159 ± 0.012 g·g-1 in stirred tank bioreactors operated in fed-batch mode. Cultivation parameters determined in fed-batch mode in bioreactors are comparable to the values obtained in chemostats, especially with respect to yields, and suggest that these findings are transferable from chemostat to fed-batch cultivations.
This thesis demonstrates the applicability and suitability of small-scale cultivations systems for the investigation of S. cerevisiae strains, purposefully engineered with synthetic biology tools to synthesize a desired product. Such production strains display a broad diversity of physiologies, requiring the systematic and solid quantification to fully exploit their potential as microbial cell factories. A small-scale system operating in fed-batch mode suitable for microtiter plate applications allows high-throughput screening approaches with production strains as well as reproducible and scalable cultivation metrics. The small-scale chemostat cultivation system enables the quantitative physiological characterization of microbial cell factories, yielding cultivation data helpful for early stages of process development. If applied appropriately, distinct features of small-scale systems, such as parallelization and high-throughput, facilitate rapid strain characterization and thus emerge to be indispensable to unleash the capabilities of microbial cell factories.