Research Synthetic Cell Factories
Group Leader: Dr. Jan Marienhagen
(Plant) Natural Product Synthesis in Microorganisms
Natural products are metabolites of the secondary metabolism that are not directly involved in growth, development or reproduction, but have important functions in defense and signaling, or serve as pigments or fragrances. Of the many hundreds of thousands natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance in the producing organisms, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most natural products. With advances in DNA sequencing and recombinant DNA technology many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways in suitable microorganisms . We apply latest molecular tools in the high-throughput format for the construction of tailor-made recombinant microorganism by metabolic engineering to modify metabolic profiles according to individual production purposes. This includes the heterologous expression of whole metabolic pathways to confer the capability for PNP synthesis, but also host cell engineering for optimized substrate utilization, improved precursor supply or reduced product degradation . This approach offers promise to provide sufficient quantities of the desired natural products from inexpensive renewable resources .
 Marienhagen J. and M. Bott (2013). Metabolic engineering of microorganisms for the synthesis of plant natural products, J. Biotechnol. 163: 166-178. http://dx.doi.org/10.1016/j.jbiotec.2012.06.001
 van Summeren-Wesenhagen P.V. and J. Marienhagen (2015). Metabolic engineering of Escherichia coli for the synthesis of the plant polyphenol pinosylvin. Appl. Environ. Microbiol. 81(3): 840-849. http://dx.doi.org/10.1128/AEM.02966-14
 van Summeren-Wesenhagen P.V. and J. Marienhagen (2013). Putting bugs to the blush: metabolic engineering for phenylpropanoid-derived products in microorganisms. Bioengineered 4: 1-8. http://dx.doi.org/10.4161/bioe.23885
Substrates from biomass for a sustainable industrial biotechnology
Methanol is already an important carbon feedstock in the chemical industry, but has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important, but non-methylotrophic bacterium Corynebacterium glutamicum towards the utilization of methanol as auxiliary carbon source in a sugar-based medium [1,2].
Biomass-derived D-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. C. glutamicum has already been engineered to grow with this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize D-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of α-ketoglutarate and its derivatives (e.g. L-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum . This five-step pathway enabled a recombinant C. glutamicum strain to utilize D-xylose in D-xylose/D-glucose mixtures and thus represents a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on D-xylose containing substrates.
 Witthoff S., Mühlroth A., Marienhagen J., Bott M. (2013) C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide. Appl. Environ. Microbiol. 79(22): 6974–6983. http://dx.doi.org/10.1128/AEM.02705-13
 Witthoff S., Schmitz K., Niedenführ S., Nöh K., Noack S., Bott M., Marienhagen J. (2015). Metabolic engineering of Corynebacterium glutamicum for the metabolization of methanol. Appl. Environ. Microbiol. 81(6): (epub ahead of print) http://dx.doi.org/10.1128/AEM.03110-14
 Radek A., Krumbach K., Gätgens J., Wendisch V. F., Wiechert W., Bott M., Noack S., Marienhagen J. (2014). Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates. J. Biotechnol. 192: 156–160.
High-throughput Metabolic Engineering of Microorganisms
Metabolic engineering for the microbial overproduction of high-value small molecules is a highly complex process, which is dictated by a large number of parameters. The biosynthetic pathways leading to these products comprise multiple native or heterologous catalytic steps, each one representing a potential bottleneck when directing carbon flux toward target small-molecule production. Furthermore, the host organism’s native genetic regulation network and the interaction with the target pathway can impact final product yields. Efforts in the field of metabolic engineering are currently defined by the implementation of a series of rational design-based strategies to modify the host genome and pathway enzymes to achieve moderate product titers. To generate microbial production strains with higher productivities, we want to follow the long trail of successes in protein engineering and develop approaches to high-throughput screening of single cells. For this purpose we construct and employ transcriptional regulators sensing target small-molecules of interest, which, in response to this signal, drive transcription of a fluorescent protein . In combination with flow cytometry these biosensors allow screening of large libraries of mutagenized single cells for improved productivities in the ultra-high-throughput format.
Furthermore, we established recombineering for genome engineering of the platform microorganism Corynebacterium glutamicum and combined this method with our biosensor-based high-throughput screening technology . We applied this new method, called RecFACS, successfully for genomic site-directed saturation mutagenesis and rapid isolation of L-lysine producing C. glutamicum cells.
 Schallmey M., Frunzke J., Eggeling L., Marienhagen J. (2014). Looking for the pick of the bunch: High-throughput screening of producing microorganisms with biosensors. Curr. Opin. Biotechnol. 26: 148-154.
 Binder S., Siedler S., Marienhagen J., Bott M., Eggeling L. (2013). Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res. 41: 6360-6369. http://dx.doi.org/10.1093/nar/gkt312
PLICing – a molecular toolbox for enzyme-free cloning, multi-site-saturation and DNA-recombination
Most methods for DNA-cloning are enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. While working at the RWTH Aachen we contributed to the development of the Phosphorothioate-based Ligase-independent Gene Cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution . PLICing starts with amplification of the target gene and the vector by PCR using primers with complementary phosphorothioated nucleotides at the 5´-end. The PCR products are cleaved in an iodine/ethanol solution, producing single-stranded overhangs. Subsequently, these ends hybridize at room temperature and the resulting DNA constructs can be directly transformed into competent host cells. PLICing is fast, simple, sequence independent and surpasses other concepts regarding cloning efficiency.
Based on the phosphorothioate-based chemistry we also developed the OmniChange method, which simultaneously and efficiently saturates five independent codons and does not require a minimal distance between mutated codons . Furthermore, the Phosphorothioate-based DNA recombination (PTRec) method allows the combinatorial recombination of structural elements or whole protein domains and only requires a short stretch of four identical amino acids among the proteins to be recombined in order to define a single crossover point . Just like PLICing, OmniChange and PTRec are technically simple, fast and robust, and should prove to be true alternatives to other well-established techniques for multi-site saturation and recombination of DNA-fragments.
 Blanusa, M., et al. (2010). Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method. Anal Biochem 406, 141-146 http://dx.doi.org/10.1016/j.ab.2010.07.011
 Dennig, A., et al. (2011). OmniChange: the sequence independent method for simultaneous site-saturation of five codons. PLoS One 6, e26222 http://dx.doi.org/10.1371/journal.pone.0026222
 Marienhagen, J., et al. (2012). Phosphorothioate-based DNA recombination: an enzyme-free method for the combinatorial assembly of multiple DNA fragments. Biotechniques 0: 1-6. http://dx.doi.org/10.2144/000113865