Biology subjects

Kourist, R.

Publications and source records attributed to Kourist, R..

3 recordsLinked to original sources

bioRxiv2024

Engineering of Transmembrane Alkane Monooxygenases to Improve a Key Reaction Step in the Synthesis of Polymer Precursor Tulipalin A

The -methylene-{gamma}-butyrolactone tulipalin A, naturally found in tulips can polymerize via addition at the vinyl group or via ring-opening polymerization, making it a highly promising monomer for biobased polymers. As tulipalin A biosynthesis in plants remains elusive, we propose a pathway for its synthesis starting from the metabolic intermediate isoprenol. For this, terminal hydroxylation of the -methylene substrate isoprenyl acetate is a decisive step. While a panel of fungal unspecific peroxygenases showed a preference for the undesired epoxidation of the exo-olefin group, bacterial alkane monooxygenases were specific for terminal hydroxylation. A combination of protein engineering based on de novo structure prediction of the membrane enzymes with cell engineering allowed to increase the specific activity by 6-fold to 1.83 U gcdw -1, unlocking this reaction for the fermentative production of tulipalin A from renewable resources.

bioengineering↗

bioRxiv2024

Strong heterologous electron sink outcompetes alternative electron transport pathways in photosynthesis

Improvement of photosynthesis requires a thorough understanding of electron partitioning under both natural and strong electron sink conditions. We applied a wide array of state-of-the-art biophysical and biochemical techniques to thoroughly investigate the fate of photosynthetic electrons in the engineered cyanobacterium Synechocystis sp. PCC 6803, a blueprint for photosynthetic biotechnology, expressing the heterologous gene for ene-reductase, YqjM. This recombinant enzyme catalyses the reduction of an exogenously added substrate into the desired product by utilising photosynthetically produced NAD(P)H, enabling whole-cell biotransformation. Through coupling the biotransformation reaction with biophysical measurements, we demonstrated that the strong artificial electron sink, outcompetes the natural electron valves, the flavodiiron protein-driven Mehler-like reaction, and cyclic electron transport. These results show that ferredoxin-NAD(P)H-oxidoreductase (FNR) is the preferred route for delivering photosynthetic electrons from reduced ferredoxin and the cellular NADPH/NADP+ ratio as a key factor in orchestrating photosynthetic electron flux. These insights are crucial for understanding molecular mechanisms of photosynthetic electron transport and harnessing photosynthesis for sustainable bioproduction by engineering the cellular source/sink balance. Furthermore, we conclude that identifying the bioenergetic bottleneck of a heterologous electron sink is a crucial prerequisite for targeted engineering of photosynthetic biotransformation platforms. Significance statementWe coupled the photosynthetic and biocatalytic (whole-cell biotransformation) performance of model cyanobacteria. We employed a heterologous NAD(P)H utilising enzyme, as a strong artificial electron sink, allowing us to gain a comprehensive understanding of photosynthetic electron partitioning. We demonstrated that the strong electron sink outcompetes natural electron sinks and cyclic electron transport.

plant biology↗

bioRxiv2021

Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 [A] resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

biophysics↗