Engineering acetyl-CoA supply and ERG9 repression to enhance mevalonate production in Saccharomyces cerevisiae.

Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis.

MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped ß subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.

Chemical investigation on the root bark of Bombax malabarica.

Ten compounds were isolated from the root bark of Bombax malabarica, including two new compounds, bombamalin (1) and 3,4,5-trimethoxyphenol-1-[ß-xylopyranosyl-(1 → 2)]-ß-glucopyranoside (3), and shorealactone (4), a rare dehydroascorbic acid fused l-ε-viniferin. Compound 1 is an unusual bissesquiterpene, containing a 1,4-dioxene ring formed by fusing isohemigossypol with ent-cadinen-2-one. Their structures were elucidated by extensive spectroscopic analysis. This chemical reinvestigation is of value for chemotaxonomy of the Bombax genus, in particular the finding of the unusual 1 and rare 4. Among the isolates, shorealactone (4), l-epicatechin 5-O-ß-D-xyloside (5), and 2-C-[ß-D-apiosyl-(1 → 6)]- ß-D-glucosyl]-1,3,6-trihydroxy-7-methoxyxanthone (6) displayed some inhibition against α-glucosidase with IC50 values of 224, 345, and 285 µM, respectively.

Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae.

BACKGROUND: Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. RESULTS: In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae. We then increased the flux through these pathways by making gene deletions of BAT1, ALD6, and PHO13, to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. CONCLUSIONS: The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

Dimerization and DNA recognition rules of mithramycin and its analogues.

The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me(2+))-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM=MTM SDK>MTM SA-Trp>MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.

Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-ß-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.

Structural insight into MtmC, a bifunctional ketoreductase-methyltransferase involved in the assembly of the mithramycin trisaccharide chain.

More and more post-PKS tailoring enzymes are recognized as being multifunctional and codependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, which-in codependence with glycosyltransferase MtmGIV-is a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by Streptomyces argillaceus. We report crystal structures of three binary complexes of MtmC with its methylation cosubstrate SAM, its coproduct SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2-2.7 Å resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is conducted by MtmC, it must bind either NADPH or SAM in the same cofactor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by ~180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open the active site for binding the bulkier cosubstrate NADPH prior to the reduction reaction.

Enzymatic methylation and structure-activity-relationship studies on polycarcin V, a gilvocarcin-type antitumor agent.

Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity relationships of the gilvocarcin group of antitumor antibiotics due to a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary O-methyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2'-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important hydrogen bond donor for the interaction with histone H3, and converting 3'-OH into an OCH3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two hydrogen bond donors in the sugar are necessary for optimal binding.

Semi-synthetic mithramycin SA derivatives with improved anticancer activity.

Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved mithramycin analogues mithramycin SK and mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to mithramycin SK. This work shows the ability to transform the previously useless mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.

Using sweeping-micellar electrokinetic chromatography to determine voriconazole in patient plasma.

Invasive fungal infection is a life-threatening condition; its occurrence has increased significantly over the past 20 years. We have developed a sensitive and efficient sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method to quantify voriconazole, a potent triazole antifungal drug, in patient plasma. Solid phase extraction (SPE) conditions were first optimized to minimize plasma interference while maintaining a high recovery; the sweeping-MEKC conditions were then systematically optimized to obtain a high sweeping efficiency with good selectivity. Under the optimal analytical conditions, voriconazole was baseline-separated from endogenous materials within 10.5 min with a limit of detection of 0.075 microg mL(-1). The background electrolyte comprised 40 mM phosphoric acid, 110 mM sodium dodecyl sulfate, and 20% acetonitrile. In terms of method repeatability, the relative standard deviations (RSDs) of the migration time and the peak area (intra-day; n=6) were both less than 5.5%; in terms of intermediate precision, and the RSDs of the peak area and the migration time (inter-day; n=3) were both less than 6.3%. We successfully applied this developed method to the quantitative determination of plasma voriconazole levels in 16 patients; the results correlated well with those obtained through analyses using high-performance liquid chromatography. This sweeping-MEKC method is accurate and efficient and appears to be applicable to therapeutic drug monitoring and clinical research.

alpha-Glucosidase inhibitors from the seeds of Syagrus romanzoffiana.

Bioassay-guided fractionation against alpha-glucosidase resulted in isolation and characterization of eight active compounds from the EtOH extract of the seeds of Syagrus romanzoffiana. Of these, seven are stilbenoids, and two of them, 13-hydroxykompasinol A (1) and scirpusin C (4), possess potent inhibitory activity against alpha-glucosidase type IV from Bacillus stearothermophilus with the IC50 value of 6.5 and 4.9 microM, respectively. The in vivo assay on normal Wistar rats using oral sucrose challenge also demonstrated that kompasinol A (2) and 3,3',4,5,5'-pentahydroxy-trans-stilbene (5) possess significant effect in reducing the postprandial blood glucose level (10.2% and 12.1% at 10mg/kg, respectively). These results suggest that stilbenoids might be explored for their therapeutic potential as hypoglycemic agents.