Mutagenesis of Dimer Interfacial Residues Improves the Activity and Specificity of Methyltransferase for cis-α-Irone Biosynthesis.

Promiscuous enzymes show great potential to establish new-to-nature pathways and expand chemical diversity. Enzyme engineering strategies are often employed to tailor such enzymes to improve their activity or specificity. It is paramount to identify the target residues to be mutated. Here, by exploring the inactivation mechanism with the aid of mass spectrometry, we have identified and mutated critical residues at the dimer interface region of the promiscuous methyltransferase (pMT) that converts psi-ionone to irone. The optimized pMT12 mutant showed ∼1.6-4.8-fold higher kcat than the previously reported best mutant, pMT10, and increased the cis-α-irone percentage from ∼70 to ∼83%. By one-step biotransformation, ∼121.8 mg L-1 cis-α-irone was produced from psi-ionone by the pMT12 mutant. The study offers new opportunities to engineer enzymes with enhanced activity and specificity.

Structural Understanding of Fungal Terpene Synthases for the Formation of Linear or Cyclic Terpene Products.

Terpene synthases (TPSs), known gatekeepers of terpenoid diversity, are the main targets for enzyme engineering attempts. To this end, we have determined the crystal structure of Agrocybe pediades linalool synthase (Ap.LS), which has been recently reported to be 44-fold and 287-fold more efficient than bacterial and plant counterparts, respectively. Structure-based molecular modeling followed by in vivo as well as in vitro tests confirmed that the region of 60-69aa and Tyr299 (adjacent to the motif "WxxxxxRY") are essential for maintaining Ap.LS specificity toward a short-chain (C10) acyclic product. Ap.LS Y299 mutants (Y299A, Y299C, Y299G, Y299Q, and Y299S) yielded long-chain (C15) linear or cyclic products. Molecular modeling based on the Ap.LS crystal structure indicated that farnesyl pyrophosphate in the binding pocket of Ap.LS Y299A has less torsion strain energy compared to the wild-type Ap.LS, which can be partially attributed to the larger space in Ap.LS Y299A for better accommodation of the longer chain (C15). Linalool/nerolidol synthase Y298 and humulene synthase Y302 mutations also produced C15 cyclic products similar to Ap.LS Y299 mutants. Beyond the three enzymes, our analysis confirmed that most microbial TPSs have asparagine at the position and produce mainly cyclized products (δ-cadinene, 1,8-cineole, epi-cubebol, germacrene D, ß-barbatene, etc.). In contrast, those producing linear products (linalool and nerolidol) typically have a bulky tyrosine. The structural and functional analysis of an exceptionally selective linalool synthase, Ap.LS, presented in this work provides insights into factors that govern chain length (C10 or C15), water incorporation, and cyclization (cyclic vs acyclic) of terpenoid biosynthesis.

A Spirobicyclo[3.1.0]Terpene from the Investigation of Sesquiterpene Synthases from Lactarius deliciosus.

Milk cap mushrooms in the genus Lactarius are known to produce a wide variety of terpene natural products. However, their repertoire of terpene biosynthetic enzymes has not been fully explored. In this study, several candidate sesquiterpene synthases were identified from the genome of the saffron milk cap mushroom L. deliciosus and expressed in a sesquiterpene-overproducing Escherichia coli strain. In addition to enzymes that produce several known terpenes, we identified an enzyme belonging to a previously unknown clade of sesquiterpene synthases that produces a terpene with a unique spiro-tricyclic scaffold. These findings add to the rich diversity of terpene scaffolds and mushroom terpene synthases and are valuable for biotechnological applications in producing these terpenoids.

Total enzymatic synthesis of cis-α-irone from a simple carbon source.

Metabolic engineering has become an attractive method for the efficient production of natural products. However, one important pre-requisite is to establish the biosynthetic pathways. Many commercially interesting molecules cannot be biosynthesized as their native biochemical pathways are not fully elucidated. Cis-α-irone, a top-end perfumery molecule, is an example. Retrobiosynthetic pathway design by employing promiscuous enzymes provides an alternative solution to this challenge. In this work, we design a synthetic pathway to produce cis-α-irone with a promiscuous methyltransferase (pMT). Using structure-guided enzyme engineering strategies, we improve pMT activity and specificity towards cis-α-irone by >10,000-fold and >1000-fold, respectively. By incorporating the optimized methyltransferase into our engineered microbial cells, ~86 mg l-1 cis-α-irone is produced from glucose in a 5 l bioreactor. Our work illustrates that integrated retrobiosynthetic pathway design and enzyme engineering can offer opportunities to expand the scope of natural molecules that can be biosynthesized.

Bioinformatics-aided identification, characterization and applications of mushroom linalool synthases.

Enzymes empower chemical industries and are the keystone for metabolic engineering. For example, linalool synthases are indispensable for the biosynthesis of linalool, an important fragrance used in 60-80% cosmetic and personal care products. However, plant linalool synthases have low activities while expressed in microbes. Aided by bioinformatics analysis, four linalool/nerolidol synthases (LNSs) from various Agaricomycetes were accurately predicted and validated experimentally. Furthermore, we discovered a linalool synthase (Ap.LS) with exceptionally high levels of selectivity and activity from Agrocybe pediades, ideal for linalool bioproduction. It effectively converted glucose into enantiopure (R)-linalool in Escherichia coli, 44-fold and 287-fold more efficient than its bacterial and plant counterparts, respectively. Phylogenetic analysis indicated the divergent evolution paths for plant, bacterial and fungal linalool synthases. More critically, structural comparison provided catalytic insights into Ap.LS superior specificity and activity, and mutational experiments validated the key residues responsible for the specificity.

Biotechnological applications of S-adenosyl-methionine-dependent methyltransferases for natural products biosynthesis and diversification.

In the biosynthesis of natural products, methylation is a common and essential transformation to alter molecules' bioavailability and bioactivity. The main methylation reaction is performed by S-adenosylmethionine (SAM)-dependent methyltransferases (MTs). With advancements in genomic and chemical profiling technologies, novel MTs have been discovered to accept complex substrates and synthesize industrially valuable natural products. However, to achieve a high yield of small molecules in microbial hosts, many methyltransferase activities have been reported to be insufficient. Moreover, inadequate co-factor supplies and feedback inhibition of the by-product, S-adenosylhomocysteine (SAH), further limit MTs' activities. Here, we review recent advances in SAM-dependent MTs to produce and diversify natural products. First, we surveyed recently identified novel methyltransferases in natural product biosynthesis. Second, we summarized enzyme engineering strategies to improve methyltransferase activity, with a particular focus on high-throughput assay design and application. Finally, we reviewed innovations in co-factor regeneration and diversification, both in vitro and in vivo. Noteworthily, many MTs are able to accept multiple structurally similar substrates. Such promiscuous methyltransferases are versatile and can be tailored to design de novo pathways to produce molecules whose biosynthetic pathway is unknown or non-existent in nature, thus broadening the scope of biosynthesized functional molecules.