Total synthesis of spiruchostatin B aided by an automated synthesizer.

The total synthesis of a natural product HDAC inhibitor, spiruchostatin B, was successfully achieved. A 5-step synthesis that included an asymmetric aldol reaction was carried out in an automated synthesizer to provide an (E)-(S)-3-hydroxy-7-thio-4-heptenoic acid segment that is the crucial structure of cysteine-containing, depsipeptidic natural products such as spiruchostatins, FK228, FR901375, and largazole for their inhibitory activity against HDACs.

Development and application of a solution-phase automated synthesizer, 'ChemKonzert'.

In this paper we describe the development of a fully-automated solution-phase synthesizer, 'ChemKonzert' that can be used to prepare a wide variety of organic compounds. The automated synthesizer is ingeniously designed to perform most of the chemical reactions currently used by synthetic organic chemists and utilizes a centrifugal separator to efficiently achieve liquid-liquid extraction. The design of the hardware and software will be described in this paper, and several examples of organic reactions will also be presented as applications of the apparatus.

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.

BACKGROUND: (+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli. RESULTS: Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. CONCLUSION: This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.

Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes.

We report here some efficient biotransformations using Escherichia coli strains with disruptions for the AcrAB-TolC efflux pump system. Biotransformations of compactin into pravastatin (6alpha-hydroxy-iso-compactin) were performed using E. coli strains with tolC and/or acrAB mutations expressing a cytochrome P450 (P450) gene. The production levels of pravastatin using strains with acrAB, tolC, and tolC acrAB mutations increased by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the production levels of 25-hydroxy vitamin D3 and 25-hydroxy 4-cholesten 3-one using tolC acrAB mutant strains expressing an individual P450 gene increased by 2.2- and 16-fold, respectively. The enhancement of this biotransformation efficiency could be explained by increases in the intracellular amounts of substrates and the concentrations of active P450s. These results demonstrate that we have achieved versatile methods for efficient biotransformations using E. coli strains with tolC acrAB mutations expressing P450 genes.

Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107.

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.

One-pot fermentation of pladienolide D by Streptomyces platensis expressing a heterologous cytochrome P450 gene.

Pladienolide D is a 16-hydroxylated derivative of pladienolide B, produced by Streptomyces platensis. To facilitate the production of pladienolide D, the gene encoding a pladienolide B 16-hydroxylase from S. bungoensis was introduced into S. platensis. The recombinant produced pladienolide D at a production level comparable to that of pladienolide B.

Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene.

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.

Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system.

Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2alpha-, 2beta-, 6beta-, 7beta-, 11beta-, 12beta-, 15beta-, 16alpha-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2alpha-, 2beta-, 6beta-, 11beta-, 15beta-, 16alpha-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the beta face.