Facile analysis of rice bran oil to compare free unsaturated fatty acid compositions of parental and hybrid rice lines.

Rice bran oil (RBO) has been a popular choice of cooking oil in several Asian countries for decades, and the interest in RBO is fast growing in Western countries due to the high levels of hearty unsaturated fats and other components beneficial to health. Further knowledge of unsaturated fatty acid content and composition in rice lines will assist in improving the quality of rice bran processing by allowing robust extraction of rice bran for oil production. The studies focused on the RBO composition of rice lines with beneficial genotypes are scarce. Accordingly, we investigated the total bran lipid content and composition of three of the most abundant, healthy, unsaturated fatty acids that freely exist in RBO: oleic, linoleic, and α-linolenic acids in nine parental lines (two male sterile lines and seven male lines) and seven hybrid rice lines, by utilizing an efficacious organic extraction to collect RBO and by developing a user-friendly reverse-phase high-performance liquid chromatography (HPLC) methodology. Our results showed that the hybrid lines had the highest oil content (F ratio = 7.2017, p value = 0.0019), while the male lines had the highest levels of two of the three free unsaturated fatty acids analyzed (linoleic acid, x ¯ = 212.801 mg and oleic acid, x ¯ = 48.132 mg). Oil weight was negatively correlated with α-linolenic acid (r = -0.6535, p value <0.0001). All three free unsaturated fatty acids were positively correlated. Our samples' natural variation in lipid content suggests that some rice lines are more suitable for oil production.

Generating single-stranded DNA circles with minimal resources.

Single-stranded circular oligonucleotides are heavily utilized in rolling circle amplification and rolling circle transcription technologies. Although various reported methodologies are available to synthesize circular, single-stranded DNA (ssDNA), the unduly complicated protocols and the associated cost minimize the utility of these methodologies to a non-expert or a beginner in the field. Our protocol provides the simplest yet robust synthesis of circular ssDNA templates to be utilized in various applications, using minimal resources.•In this manuscript, we describe the most basic approach to synthesize circular ssDNA.•Our method utilizes the minimal resources, yet it is robust.•The utility of the methodology is very high for a non-expert or a beginner in the field.

CoA recycling by a benzoate coenzyme A ligase in cascade reactions with aroyltransferases to biocatalyze paclitaxel analogs.

A Pseudomonas CoA ligase (BadA) biocatalyzed aroyl CoA thioesters used by a downstream N-benzoyltransferase (NDTNBT) in a cascade reaction made aroyl analogs of the anticancer drug paclitaxel. BadA kept the high-cost aroyl CoA substrates at saturation for the downstream NDTNBT by recycling CoA when it was added as the limiting reactant. A deacylated taxane substrate N-debenzoyl-2'-deoxypaclitaxel was converted to its benzoylated product at a higher yield, compared to the converted yield in assays in which the BadA ligase chemistry was omitted, and benzoyl CoA was added as a cosubstrate. The resulting benzoylated product 2'-deoxypaclitaxel was made at 196% over the theoretical yield of product that could be made from the CoA added at 50 µM, and the cosubstrates benzoic acid (100 µM), and N-debenzoyl-2'-deoxypaclitaxel (500 µM) added in excess. In addition, a 2-O-benzoyltransferase (mTBT) was incubated with BadA, aroyl acids, CoA, a 2-O-debenzoylated taxane substrate, and cofactors under the CoA-recycling conditions established for the NDTNBT/BadA cascade. The mTBT/BadA combination also made various 2-O-aroylated products that could potentially function as next-generation baccatin III compounds. These ligase/benzoyltransferase cascade reactions show the feasibility of recycling aroyl CoA thioesters in vitro to make bioactive acyl analogs of paclitaxel precursors.

X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins.

Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the ß subunit, blocking the path of the RNA transcript. The C3'-tail of benzoxazinorifamycin fits a cavity between the ß subunit and σ factor. We propose that in addition to blocking RNA exit, the benzoxazinorifamycin C3'-tail changes the σ region 3.2 loop position, which influences the template DNA at the active site, thereby reducing the efficiency of transcription initiation. This study supports expansion of structure-activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.

Point mutations (Q19P and N23K) increase the operational solubility of a 2alpha-O-benzoyltransferase that conveys various acyl groups from CoA to a taxane acceptor.

Two site-directed mutations within the wild-type 2-O-benzoyltransferase (tbt) cDNA, from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed the conversion of each 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III to several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues. The relative catalytic efficiency of mTBT with the 7,13-O,O-diacetyl-2-O-debenzoyl surrogate substrate and heterole carbonyl CoA substrates was slightly greater than with the natural aroyl substrate benzoyl CoA, while substituted benzoyl CoA thioesters were less productive. Short-chain hydrocarbon carbonyl and cyclohexanoyl CoA thioesters were also productive, where C(4) substrates were transferred by mTBT with approximately 10- to 17-fold greater catalytic efficiency compared to the transfer of benzoyl. The described broad specificity of mTBT suggests that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.

An N-aroyltransferase of the BAHD superfamily has broad aroyl CoA specificity in vitro with analogues of N-dearoylpaclitaxel.

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.