Nepeta cataria L. (catnip) can serve as a chassis for the engineering of secondary metabolic pathways.

OBJECTIVE: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences. RESULTS: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 µmol g-1 s-1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 µmol g-1 s-1 and 18 ± 4 µmol g-1 s-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides. CONCLUSION: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity.

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.

Winning the numbers game in enzyme evolution - fast screening methods for improved biotechnology proteins.

The booming demand for environmentally benign industrial processes relies on the ability to quickly find or engineer a biocatalyst suitable to ideal process conditions. Both metagenomic approaches and directed evolution involve the screening of huge libraries of protein variants, which can only be managed reasonably by flexible platforms for (ultra)high-throughput profiling against the desired criteria. Here, we review the most recent additions toward a growing toolbox of versatile assays using fluorescence, absorbance and mass spectrometry readouts. While conventional solution based high-throughput screening in microtiter plate formats is still important, the implementation of novel screening protocols for microfluidic cell or droplet sorting systems supports technological advances for ultra-high-frequency screening that now can dramatically reduce the timescale of engineering projects. We discuss practical issues of scope, scalability, sensitivity and stereoselectivity for the improvement of biotechnologically relevant enzymes from different classes.

Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4.

Human drug-metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)-epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.

Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds.

Employment of transient expression of foreign genes for bioconversion of pharmaceutically valuable low-molecular-weight compounds, including plant secondary metabolites, is an enticing trend still scantily explored in plant biotechnology. In the present work, an efficient protocol for rapid assessment of synthetic and plant-derived metabolites as potential substrates for human P450s (CYP2D6 and CYP3A4) via Agrobacterium-mediated transient expression in Nicotiana benthamiana is put forth. Animal P450s with broad substrate specificity are promising candidates for transformation of diverse metabolites. The efficiency of P450s in heterologous surroundings is not always satisfactory and depends on the availability of an associated electron-transfer enzyme. Plants represent an attractive assortment of prospective hosts for foreign P450s expression. The optimal composition of genetic blocks providing the highest transient expression efficiency is designed, an effective substrate administration scheme is validated, and biological activity of the investigated P450s against loratadine and several indole alkaloids with different molecular scaffold structures is tested. A novel indole alkaloid, 11-hydroxycorynanthine, is isolated from N. benthamiana plants transiently expressing CYP2D6 and supplemented with corynanthine, and its structure was elucidated. The proposed technique might be of value in realization of combinatorial biosynthesis concept comprising the junction of heterologous enzymes and substrates in different metabolic surroundings.

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

Agrobacterium-mediated transient expression as an approach to production of recombinant proteins in plants.

Agrobacterium-mediated transient expression is a recently developed approach to quick and inexpensive large-scale production of recombinant proteins in plant systems. In the course of this process, foreign gene expression occurs during several days after agroinfiltration without integration of recombinant DNA into plant genome and the level of target protein synthesis may be considerably higher than in stably transformed plants. This mini-review describes biological peculiarities of transient expression process, method development and optimization, and the range of application for biotechnological production of variable proteins. Patenting the transient expression protocols that mostly occurred during new millennium indicates the growing interest to this method as a perspective alternative to "classical" pro- and eukaryotic systems for recombinant protein production.