Enlighting Electron Routes In Oxyfunctionalizing Synechocystis sp. PCC 6803.

Phototrophic microorganisms, like cyanobacteria, are gaining attention as host organisms for biocatalytic processes with light as energy source and water as electron source. Redox enzymes, especially oxygenases, can profit from in-situ supply of co-substrates, i. e., reduction equivalents and O2 , by the photosynthetic light reaction. The electron transfer downstream of PS I to heterologous electron consuming enzymes in principle can involve NADPH, NADH, and/or ferredoxin, whereas most direct and efficient transfer is desirable. Here, we use the model organism Synechocystis sp. PCC 6803 to investigate, to what extent host and/or heterologous constituents are involved in electron transfer to a heterologous cytochrome P450 monooxygenase from Acidovorax sp. CHX100. Interestingly, in this highly active light-fueled cycloalkane hydroxylating biocatalyst, host-intrinsic enzymes were found capable of completely substituting the function of the Acidovorax ferredoxin reductase. To a certain extent (20 %), this also was true for the Acidovorax ferredoxin. These results indicate the presence of a versatile set of electron carriers in cyanobacteria, enabling efficient and direct coupling of electron consuming reactions to photosynthetic water oxidation. This will both simplify and promote the use of phototrophic microorganisms for sustainable production processes.

Identification of three novel P450 enzymes involved in the oxidative modification of a newly discovered fusicoccane diterpene.

Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5-8-5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2-5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5-8-5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis.

Discovery of sesquiterpenoids from an actinomycete Crossiella cryophila through genome mining and heterologous expression.

Genome mining of the Actinomycete Crossiella cryophila facilitated the discovery of a minimal terpenoid biosynthetic gene cluster of cry consisting of a class I terpene cyclase CryA and a CYP450 monooxygenase CryB. Heterologous expression of cry allowed the isolation and characterization of two new sesquiterpenoids, ent-viridiflorol (1) and cryophilain (2). Notably, cryophilain (2) possesses a 5/7/3-fused tricyclic skeleton bearing a distinctive bridgehead hydroxy group. The combined in vivo and in vitro experiments revealed that CryA, the first ent-viridiflorol terpene cyclase, catalyzes farnesyl diphosphate to form the 5/7/3 sesquiterpene core scaffold and P450 CryB serves as a tailoring enzyme responsible for installing a hydroxy group at the bridgehead carbon.

OsCPD1 and OsCPD2 are functional brassinosteroid biosynthesis genes in rice.

CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), member of the CYP90A family of cytochrome P450 (CYP450) monooxygenase, is an essential component of brassinosteroids (BRs) biosynthesis pathway. Compared with a single CPD/CYP90A1 in Arabidopsis thaliana, two highly homologous CPD genes, OsCPD1/CYP90A3 and OsCPD2/CYP90A4, are present in rice genome. There is still no genetic evidence so far about the requirement of OsCPD1 and OsCPD2 in rice BR biosynthesis. In this study, we reported the functional characterization of OsCPD genes using CRISPR/Cas9 gene editing technology. The overall growth and development of oscpd1 and oscpd2 single knock-out mutants was indistinguishable from the wild-type, whereas, the oscpd1 oscpd2 double mutant displayed multiple and obvious BR-related defects. Cytological analyses further indicated the defective cell elongation in oscpd1 oscpd2 double mutant. The oscpd double mutants had a lower endogenous BR level and could be restored by the application of the brassinolide (BL). Moreover, overexpression of OsCPD1 and OsCPD2 led to a typical BR enhanced phenotype, with enlarged leaf angle and increased grain size. Taken together, our results provide direct genetic evidence that OsCPD1 and OsCPD2 play essential and redundant roles in maintenance of plant architecture by modulating BR biosynthesis in rice.

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120.

Cytochrome P450 monooxygenases (Cyps) effectively catalyze the regiospecific oxyfunctionalization of inert C-H bonds under mild conditions. Due to their cofactor dependency and instability in isolated form, oxygenases are preferably applied in living microbial cells with Pseudomonas strains constituting potent host organisms for Cyps. This study presents a holistic genetic engineering approach, considering gene dosage, transcriptional, and translational levels, to engineer an effective Cyp-based whole-cell biocatalyst, building on recombinant Pseudomonas taiwanensis VLB120 for cyclohexane hydroxylation. A lac-based regulation system turned out to be favorable in terms of orthogonality to the host regulatory network and enabled a remarkable specific whole-cell activity of 34 U gCDW -1. The evaluation of different ribosomal binding sites (RBSs) revealed that a moderate translation rate was favorable in terms of the specific activity. An increase in gene dosage did only slightly elevate the hydroxylation activity, but severely impaired growth and resulted in a large fraction of inactive Cyp. Finally, the introduction of a terminator reduced leakiness. The optimized strain P. taiwanensis VLB120 pSEVA_Cyp allowed for a hydroxylation activity of 55 U gCDW -1. Applying 5 mM cyclohexane, molar conversion and biomass-specific yields of 82.5% and 2.46 mmolcyclohexanol gbiomass -1 were achieved, respectively. The strain now serves as a platform to design in vivo cascades and bioprocesses for the production of polymer building blocks such as ε-caprolactone.