Biology subjects

Malihan-Yap, L.

Publications and source records attributed to Malihan-Yap, L..

3 recordsLinked to original sources

bioRxiv2025

Engineering Membrane-Bound Alkane Monooxygenase from Marinobacter sp. for Increased Activity in the Selective ω-Hydroxylation of Linear and Branched Aliphatic Esters

The regio- and stereoselective hydroxylation of unactivated C(sp3)-H bonds is an important reaction in organic synthesis. While bacterial alkane monooxygenase AlkB catalyzes the terminal hydroxylation of aliphatic esters with excellent regioselectivity, the molecular principles of substrate recognition and selectivity of this integral membrane enzyme are still poorly understood. In this study, we investigated the substrate scope and engineered the medium-chain alkane monooxygenase from Marinobacter sp. (M_AlkB) for the terminal hydroxylation of linear and branched esters of fatty acids and alcohols. For the first time, we demonstrated the stereoselectivity of AlkB towards prochiral substrates containing terminal gem-dimethyl groups, leading to the corresponding chiral {beta}-methyl primary alcohols in good optical purity (51-79% ee). The hydroxylation products can be further derivatized to chiral diols and lactones. Substitution of the highly conserved active site residue F169 to leucine increased the activity towards short and medium-chain esters up to two-fold. While the wildtype enzyme does not accept long-chain substrates, activity towards n-dodecyl acetate could be unlocked by reducing the size of the tryptophan residue 60 situated in the putative substrate tunnel. Substitution of the peripheral I238 with valine increased activity regardless of the chain length of the substrate. Our results lay the groundwork for the establishment of a whole-cell process for the regio- and stereoselective hydroxylation of linear and branched esters, leading to valuable bifunctionalized products. The insights gained from mutating key residues and the substrate acceptance of AlkB will guide future protein engineering campaigns. SIGNIFICANCEWe report rational engineering of a membrane-bound alkane monooxygenase in the hydroxylation of aliphatic esters, giving rise to diols, hydroxy acids, and lactones. By site-directed mutagenesis, we increased the activity towards short and medium-chain substrates, and we unlocked the activity towards long-chain substrates. We uncovered the enzymes capacity to discriminate between prochiral methyl groups, establishing new routes for the synthesis of chiral diols and lactones, important building blocks for the pharmaceutical industry.

biochemistry↗

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↗