Compartmentalized biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and ß-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB' as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC' and O-methyltransferase MpaG', the Golgi apparatus-associated prenyltransferase MpaA', the endoplasmic reticulum-bound oxygenase MpaB' and P450-hydrolase fusion enzyme MpaDE', and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH'. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal ß-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.

Functional characterization of MpaG', the O-methyltransferase involved in the biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA, 1) is a clinically important immunosuppressant. In this report, a gene cluster mpa' responsible for the biosynthesis of 1 was identified from Penicillium brevicompactum NRRL 864. The S-adenosyl-L-methionine-dependent (SAM-dependent) O-methyltransferase encoded by the mpaG' gene was functionally and kinetically characterized in vitro. MpaG' catalyzes the methylation of demethylmycophenolic acid (DMMPA, 6) to form 1. It also showed significant substrate flexibility by methylating two structural derivatives of 6 prepared by organic synthesis.

New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

Analysis of ΔpH and the xanthophyll cycle in NPQ of the Antarctic sea ice alga Chlamydomonas sp. ICE-L.

Non-photochemical fluorescence quenching (NPQ) is mainly associated with the transthylakoid proton gradient (ΔpH) and xanthophyll cycle. However, the exact mechanism of NPQ is different in different oxygenic photosynthetic organisms. In this study, several inhibitors were used to study NPQ kinetics in the sea ice alga Chlamydomonas sp. ICE-L and to determine the functions of ΔpH and the xanthophyll cycle in the NPQ process. NH4Cl and nigericin, uncouplers of ΔpH, inhibited NPQ completely and zeaxanthin (Z) was not detected in 1 mM NH4Cl-treated samples. Moreover, Z and NPQ were increased in the samples containing N,N'-dicyclohexyl-carbodiimide (DCCD) under low light conditions. We conclude that ΔpH plays a major role in NPQ, and activation of the xanthophyll cycle is related to ΔpH. In dithiothreitol (DTT)-treated samples, no Z was observed and NPQ decreased. NPQ was completely inhibited when NH4Cl was added suggesting that part of the NPQ process is related to the xanthophyll cycle and the remainder depends on ΔpH. Moreover, lutein and ß-carotene were also essential for NPQ. These results indicate that NPQ in the sea ice alga Chlamydomonas sp. ICE-L is mainly dependent on ΔpH which affects the protonation of PSII proteins and de-epoxidation of the xanthophyll cycle, and the transthylakoid proton gradient alone can induce NPQ.

Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.

Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis.

A transthylakoid proton gradient and inhibitors induce a non-photochemical fluorescence quenching in unicellular algae Nannochloropsis sp.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. In this report, special chemicals were used to perturb the kinetics of the ΔpH build-up and the xanthophyll cycle (XC) in Nannochloropsis sp. We found that NPQ was stimulated rapidly on exposure to high light and relaxed rapidly in darkness. The ΔpH could be obligatory for NPQ and ΔpH alone was not sufficient to induce NPQ. The XC, being strictly mediated by ΔpH, was also essential for NPQ. The results demonstrate that the mechanism of NPQ in Nannochloropsis sp. resembled that of diatoms.

Temperature regulates fatty acid desaturases at a transcriptional level and modulates the fatty acid profile in the Antarctic microalga Chlamydomonas sp. ICE-L.

Chlamydomonas sp. ICE-L which can thrive in extreme environments of the Antarctic is a major biomass producer. The FAD genes in Chlamydomonas sp. ICE-L were obtained and sequence alignment showed that these genes are homologous to known FADs with conserved histidine motifs. In this study, we analyzed the transcription of five FADs and FA compositions at different temperatures. The results showed that the expressions of Δ9CiFAD, ω3CiFAD1 and ω3CiFAD2 were apparently up-regulated at 0°C, however, the up-regulation of Δ6CiFAD intensified with rising temperature. Meanwhile, analysis of the FA compositions showed that PUFAs were dominant compositions, accounting for more than 75% TFA in Chlamydomonas sp. ICE-L. Furthermore, PUFAs were significantly increased at 0 and 5°C, which may be attributed to higher proportions of C18:3 and C20:3. Moreover, PUFAs were significantly decreased at 15°C whereas SFAs were significantly increased.

Complete mitochondrial genome of the sea slug, Placida sp., contains unusual noncoding regions.

The complete sequence (14,751 bp) of the mitochondrial DNA (mtDNA) of the opisthobranch gastropod Placida sp. was determined using long PCR and genome-walking techniques. The genome contains 13 protein-coding genes (PCG), 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes that are typical of metazoan mtDNA, and 2 lengthy noncoding regions with a total length of 1441 bp. All the tRNA genes have general secondary structures without reduced T or D stems. The mitochondrial gene arrangement of Placida sp. is almost identical to that of sea slugs, whereas the tRNA gene arrangement is different from other animals outside Sacoglossa in the Euthyneura. A phylogenetic analysis based on the mitochondrial genome of Euthyneura indicated that Placida sp. and Placida dendritica are closely related to Elysia chlorotica as a sister taxon, which strongly supports their close phylogenetic affinity.

Evidence of coexistence of C3 and C4 photosynthetic pathways in a green-tide-forming alga, Ulva prolifera.

Ulva prolifera, a typical green-tide-forming alga, can accumulate a large biomass in a relatively short time period, suggesting that photosynthesis in this organism, particularly its carbon fixation pathway, must be very efficient. Green algae are known to generally perform C3 photosynthesis, but recent metabolic labeling and genome sequencing data suggest that they may also perform C4 photosynthesis, so C4 photosynthesis might be more wide-spread than previously anticipated. Both C3 and C4 photosynthesis genes were found in U. prolifera by transcriptome sequencing. We also discovered the key enzymes of C4 metabolism based on functional analysis, such as pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK). To investigate whether the alga operates a C4-like pathway, the expression of rbcL and PPDK and their enzyme activities were measured under various forms and intensities of stress (differing levels of salinity, light intensity, and temperature). The expression of rbcL and PPDK and their enzyme activities were higher under adverse circumstances. However, under conditions of desiccation, the expression of rbcL and ribulose-1, 5-biphosphate carboxylase (RuBPCase) activity was lower, whereas that of PPDK was higher. These results suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C4-type carbon metabolism in U. prolifera, probably contributing to its wide distribution and massive, repeated blooms in the Yellow Sea.