Fermentative production of monomeric phenylpropanoids

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Sustainable synthesis of valuable chemicals is often dependent on plant natural products such as the phenylpropanoids. The building blocks are mainly derived from decomposition of plant material, a costly and energy-demanding process. With the advance of genetic engineering of microbes, new routes have emerged relying on de novo biosynthesis in microbial hosts. However, these processes need years of bioengineering to obtain high product yields. Such bottom-up synthesis of monomeric phenylpropanoids as building blocks for the chemical industry has recently become achievable by establishing the initial steps of a synthetic pathway in microbes. The yield and diversity of products obtained are, however, still lagging behind the natural biosynthetic pathways found in plants. The project aims to expand the power of this synthetic pathway by engineering approaches on various levels: the availability of precursors, the efficiency of individual catalytic steps and the diversification of the product range. Since the established pathway depends on the availability of L-tyrosine, microbial hosts will be screened for improved performance on species and strain level, in particular strains of the traditional workhorse for industrial amino acid production, Corynebacterium glutamicum, designed to overproduce aromatic amino acids. The efficiency of the following catalytic steps is then going to be enhanced by screening for alternative enzymes and enzyme constructs. In particular for the Cytochrome P450-catalyzed step, modular tethering strategies with interacting redox enzymes will be developed that can also be applied to other pathways and thus provide means to overcome low enzyme activity, often a major hurdle in pathway design. In addition, downstream processing of the monomeric precursors by methylation will be established to finally provide a broad range of natural monolignols via microbial fermentation. Therefore, a range of O-methyltransferases from various organisms is going to be functionally characterized to gain insight into this crucial product tailoring step for pathway engineering projects in general and the phenylpropanoid pathway in particular. Lastly, a CRISPR/Cas9-based genome-editing system will be developed for C. glutamicum to facilitate the stable integration of pathways into this model organism and to increase its value for pathway engineering. The proposed study will not only increase the efficiency of the synthetic phenylpropanoid pathway but provide tools to further our abilities in manipulating microbial hosts for the fermentative production of valuable chemicals.