High throughput cultivation systems are an essential step to increase the speed during the development of bioprocesses, e.g., the production of drugs in microorganisms (Neubauer et al., 2013). High numbers of possible combinations of strains and cultivations conditions arise during screening for optimal producers, and miniaturization is a key factor in making these large number of experiments economically and practically feasible (Long et al., 2014). High throughput cultivation systems have evolved over the years with many options for monitoring and manipulation of the cultivation conditions (Hemmerich et al., 2018; Teworte et al., 2022).

However, further challenges must be tackled to mimic the production conditions more closely and thus minimize scale-up risks. Industrial bioprocesses are typically highly dynamic and stress factors such as heterogeneities inside the reactor may lead to metabolic changes (Lin & Neubauer, 2000). Further difficulties lie in limitations in real-time monitoring and the need to handle high numbers of parallel cultivations at very small volumes.

My group tackles those difficulties applying model-based methods. Mathematical models allow us to extract more information from our experimental data, and to optimize the production of the desired active ingredients. Optimal growth conditions for the microbial host, usually Escherichia coli, can be explored in silico, and critical process variables such as the biomass concentration are monitored, e.g., by state estimation using the scarce available online measurements. Based on the detected dynamics, the feed rate is iteratively adapted to avoid violation of cultivation constraints, e.g., overflow metabolism or oxygen limitation. Furthermore, we study the impact of heterogeneities, e.g., oscillations in the glucose concentration, on growth and production by applying different cultivations modes. Altogether, our work aims to study large scale phenomena in small-scale systems, and thus aids in the development of robust strains.

Ultraviolet radiation (UV; 290-400 nm) comprises a relatively small fraction of solar radiation that reaches the Earth's surface. High-energy shorter wavelengths of solar UV (UVB; 290-315 nm) can potentially cause a range of adverse effects in plants, including disruption of the integrity and function of important macromolecules (DNA, proteins, and lipids), oxidative damage, changes in biochemistry, partial inhibition of photosynthesis, and growth reduction.¹ Consequently, UV-B has traditionally been considered a stressor. However, recent studies have highlighted the regulatory properties of low, ecologically relevant doses of UV-B, which trigger significant changes in plant secondary metabolism.

Flavonoid glycosides and hydroxycinnamic acid derivatives are ubiquitously found in plants. They are antioxidants in planta and in humans and therefore interesting compounds for the production of functional food. Along the value chain from cultivar selection through pre- and postharvest, there is significant potential for the production of functional foods with elevated concentrations of flavonoid glycosides and hydroxycinnamic acid derivatives.

UV-B is one of the most effective abiotic factors to promote the biosynthesis of flavonoid glycosides and hydroxycinnamic acid derivatives with high antioxidant activity. It has been shown that chemical-structural properties of flavonoid glycosides and hydroxycinnamic acid derivatives strongly influence antioxidant activity.² The new possibility of narrow-band application of UV-A and UV-B using LEDs provides an innovative approach to target enhancement of flavonoid glycosides and hydroxycinnamic acid derivatives with high antioxidant activity in food plants. As expected, the effect of UV-B compared to two wavelengths from the UV-A spectrum resulted in the highest induction of flavonoid glycosides, but not of hydroxycinnamic acid derivatives.³ With regard to the production of functional food, UV radiation is a particularly effective factor for influencing the phenolic profile in food plants.

For the generation of bulk chemicals to be used at a ton scale, the industry still primarily relies on petrol-based resources and classical chemical synthetic strategies, simply because economic factors outrun ecologic criteria. However, as our society strives for a more circular (bio)economy, there's a pressing need to reassess and potentially overhaul these practices in favour of more sustainable synthetic strategies.

Aliphatic compounds with terminal functionalization are ubiquitous starting materials for many materials such as polymers, cosmetics, or additives in dyes and glues. While different strategies have been devised to produce such compounds through biotechnological means, a significant gap in the mid-chain-length substrate range remains. We have tackled this issue by using oxygenases for the terminal hydroxylation of alcohols.

To this end, cytochrome P450 monooxygenases (CYPs) bear potential as extremely versatile and yet selective oxyfunctionalization catalysts. One interesting CYP subfamily is the CYP153 family. These enzymes are described as hydroxylases for medium-length alkanes, with a high regioselectivity for terminal hydroxylation of linear alkanes. This family can also use cyclic alkanes as substrates, and di-hydroxylate alkanes/alcohols and catalyze the epoxidation of alkenes.

In an efficient CYP catalysis, the electron supply plays a pivotal role. We have laid focus on a pair of Ferredoxin and Ferredoxin reductase that has been found together with a CYP153A and shows great potential for productive electron supply towards the target CYP. Furthermore, methods of creating chimeric fusion proteins give access to productive enzymes.

Different CYP153 enzymes were artificially fused to an electron transport system and produced in Escherichia coli. In whole-cell biotransformation, different compounds were used to test for reaction capacity (e.g. hydroxylation or epoxidation). Whilst colorimetric assays were used for general activity assessment, substrate and product quantification was achieved by liquid- and gas chromatographic methods. Reaction engineering addressed challenges that have to be met in a whole-cell format, as e.g. the tolerance of the host cells towards substrates and products, and methods to tackle downstream processing issues.

Substrate screening revealed a highly versatile reactivity throughout our CYP panel, of which some examples will be highlighted. From our collection of CYP153s, candidates able to perform different reactions were identified and their production optimized. Results from whole-cell biotransformations gave valuable insight into the factors attributing to reactivity and toxicity[1] of the target products.

Wax esters (WEs) are neutral lipids of major industrial importance used as components of lubricants, pharmaceuticals, and cosmetics. Large-scale production of WEs is based on chemical processes, which utilize petroleum-derived feedstocks and generate hazardous waste. Comparably small amounts of natural WEs can be obtained from Simmondsia chinensis (jojoba), the only plant known to use WEs as major storage lipids. Despite recent advances in lipid biotechnology, platforms for efficient bio-based WE production are not yet available. Metabolic engineering of oilseeds for WE accumulation represents a promising strategy for renewable, sustainable, and environment-friendly production of lipids tailored to industrial applications

The formation of WEs from acyl-CoAs and fatty alcohols requires the activity of only two enzymes: a fatty acyl reductase (FAR) and a wax synthase (WS). Numerous studies demonstrated the establishment of the WE biosynthetic pathway in seeds of non-food plants, such as Arabidopsis thaliana, Camelina sativa, and Crambe abyssinica. However, seeds with high amounts of WEs showed reduced germination rates and defects during post-germinative growth, most probably due to poor WE catabolism and/or accumulation of potentially toxic fatty alcohols [1].

In the current approaches aimed at producing WEs in oilseeds, two pathways are the main focus: WE synthesis and mobilisation. The isolation and characterisation of FARs and WSs of various origins and different specificities opened the possibility of modifying the chemical structure of the WEs accumulated in the seeds. Meanwhile, studying WE mobilisation in jojoba seeds led to identification of three enzymatic activities required in this process: a lipase, a fatty alcohol oxidase (FAO), and a fatty aldehyde dehydrogenase (FADH) [2].

During the talk, I will focus on the strategies of WE production in seeds, including the investigation of the substrate specificity of the selected FARs and WSs [3], and on characterisation of WE-hydrolysing activity of lipases from jojoba [4], C. sativa, and C. abyssinica.

One of the biggest current societal challenges is to develop a sustainable bioeconomy and reduce our dependency on finite natural resources. Cyanobacteria are ubiquitous oxygenic photosynthetic bacteria with a versatile metabolism. Some fast-growing strains have doubling times of two hours – similar to certain yeast strains. Their photosynthetic capabilities combined with a fast-growth phenotype make them attractive for biotechnology. However, technical and biological limitations will have to be overcome to achieve scalable systems.

Cyanobacteria typically are known to have a blue-green colour derived from their pigment profile of chlorophylls, carotenoids and phycobilins. Therefore, their native capacity for pigment production is a promising starting point to extend the pigment profile by genetic and metabolic engineering. In this talk, I will give an overview of current work on exploring the potential of sustainable pigment production in cyanobacteria to produce yellow and red pigments.

After the presentation, there will be a career discussion panel for early career CeBiTec scientists in G01-101.

Model organisms are essential tools for biological research. Much of our understanding of plant development, metabolism, and resistance to biotic and abiotic stresses is derived from the genetic model Arabidopsis thaliana. However, plants are a diverse lineage that have evolved suitably diverse mechanisms to adapt and thrive in every terrestrial ecosystem. The moss Physcomitrium patens is an accessible system in that it has a fully sequenced genome, and tools for its genetic manipulation have developed well in the last decade. The tractability of P. patens as a genetic model plant is outstanding, given its haploid-dominant life cycle, clonal propagation, and cellular totipotency. Further, its minimalistic organ structure and developmental patterning greatly simplify characterization of physiological and developmental processes. From this perspective, P. patens has untapped potential. We have developed tools for high-throughput CRISPR/Cas9 mutagenesis, transient expression assays, and allele replacement in P. patens, within the framework of our studies on sphingolipids, which are essential and abundant membrane components. This platform has allowed us to obtain large collections of mutant alleles with a spectrum of phenotype severities, and revealed roles for specific sphingolipid moieties in cytokinesis. Expanding basic plant research to phylodiverse model species will permit new experimental approaches, and will support applied work to understand and improve plant resilience in a changing global climate.