Historically, the majority of new drugs have been generated from natural products (secondary metabolites) and from compounds derived from natural products. During the past 15 years, pharmaceutical industry research into natural products has declined, in part because of an emphasis on high-throughput screening of synthetic libraries. Currently there is substantial decline in new drug approvals and impending loss of patent protection for important medicines. However, untapped biological resources, "smart screening" methods, robotic separation with structural analysis, metabolic engineering, and synthetic biology offer exciting technologies for new natural product drug discovery. Advances in rapid genetic sequencing, coupled with manipulation of biosynthetic pathways, may provide a vast resource for the future discovery of pharmaceutical agents.

Highly-reducing iterative polyketide synthases are large multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of LovB (the Lovastatin Nonaketide Synthase) from an engineered strain of Saccharomyces cerevisiae, and complete reconstitution of its catalytic function in the presence and absence of cofactors (NADPH, SAM) and its partner enzyme, the enoyl reductase LovC. The results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding the functions and structures of this family of enzymes.Nature uses an amazing array of enzymes to make natural products (1). Among these metabolites, polyketides represent a class of over 7000 known structures of which more than 20 are commercial drugs (2). Among the most interesting but least understood enzymes making these compounds are the highly-reducing iterative polyketide synthases (HR-IPKSs) found in filamentous fungi (3). In contrast to the well-studied bacterial type I PKSs that operate in an assembly-line fashion (4), HR-IPKSs are megasynthases that function iteratively by using a set of catalytic domains repeatedly in different combinations to produce structurally diverse fungal metabolites (5). One such metabolite is lovastatin, a cholesterol-lowering drug from Aspergillus terreus (6). This compound is a precursor to simvastatin (Zocor™), a semisynthetic drug that had annual sales of over $4.3 billion prior to loss of patent protection in 2006 (7).Biosynthesis of lovastatin proceeds via dihydromonacolin L (acid form 1; lactone form 2), a product made by the HR-IPKS, LovB (Lovastatin Nonaketide Synthase), with assistance of a separate enoyl reductase, LovC (8) (Fig. 1). LovB is a 335 kDa protein that contains single copies of ketosynthase (KS), malonyl-CoA:ACP acyltransferase (MAT), dehydratase (DH), § To whom correspondence should be addressed. yitang@ucla.edu (Y.T.); john.vederas@ualberta.ca (J.C.V.).Reconstitution of catalytic function provides insight into how multifunctional enzymes synthesize important natural products. This enzyme also catalyzes a biological Diels Alder reaction during the assembly process to form the decalin ring system (10). In vitro studies of LovB (11) have been hampered by inability to obtain sufficient amounts of the functional purified megasynthase from either A. terreus or heterologous Aspergillus hosts. As a result, the programming that governs metabolite assembly by LovB or other HR-IPKSs is not understood. Key aspects that remain to be elucidated include: 1) the catalytic and structural roles of each domain in the megasynthase; 2) substrate specificities of the catalytic domains and their tolerance to...

Drug discovery and natural products: end of era or an endless frontier? Перевод печатается с разрешения авторов и журнала Science)Исторически большинство новых лекарств разрабатывалось из природных продуктов (или их вторичных метаболитов) и из полученных из них соединений. В течение последних 15 лет исследования фармацевтической индустрии в области природных веществ заметно снизились, и одной из причин этого можно считать особое внимание к высоко производительному скринингу библиотек синтетических соединений. В настоящее время отмечается существенное снижение количества новых одобренных соответствующими инстанциями лекарств, а также потеря патентной защиты у ряда важных препаратов. Однако не иссякающие биологические источники, методы быстрого скрининга, процедуры роботизированного выделения со структурным анализом, метаболическое конструирование и синтетическая биология открывают перспективные технологические возможности для создания лекарств на основе новых природных соединений. Достижения в быстром генетическом секвенировании, в сочетании с комбинированным использованием биосинтетических путей, могут обеспечить обширный ресурс для создания новых фармакологических агентов в будущем.Ключевые слова: новые лекарства, природные соединения, библиотеки синтетических соединений, скрининг биологической активности, генетическое секвенирование, синтетическая биология.Около 200 лет назад 21-летний фармацевт Фридрих Сертюмер выделил первое биологически активное соединение из растения: морфин из опиума, вырабатываемого семенами мака, Papaver somniferum [1]. С этого момента началась эра получения из растений лекарств, которые после выделения и изучения свойств вводили в определённых дозах, не зависимых от источника или возраста материала. После Второй Мировой Войны, в связи с открытием пенициллина, фармацевтические исследования расширились, включив массивный скрининг микроорганизмов для поиска новых антибиотиков. К 1990 г. около 80% лекарств были или природными веществами, или их аналогами. Антибиотики (например, пенициллин, тетрациклин, эритромицин), антипаразитарные лекарства (авермектин), антималярийные (хинин, артемизинин), контролирующие уровень липидов агенты (ловастин и его аналоги), иммуно-супрессанты для трансплантации органов (например, циклоспорин, рапамицины), противораковые 148 * -адресат для переписки

Hypothemycin is a macrolide protein kinase inhibitor from the fungus Hypomyces subiculosus. During biosynthesis, its carbon framework is assembled by two iterative polyketide synthases (PKSs), Hpm8 (highly reducing) and Hpm3 (non-reducing). These were heterologously expressed in Saccharomyces cerevisiae BJ5464-NpgA, purified to near homogeneity and reconstituted in vitro to produce (6′S, 10′S)-trans-7′,8′-dehydrozearalenol (1) from malonyl-CoA and NADPH. The structure of 1 was determined by x-ray crystallographic analysis. In the absence of functional Hpm3, the reducing PKS Hpm8 produces and offloads truncated pyrone products instead of the expected hexaketide. The non-reducing Hpm3 is able to accept an N-acetylcysteamine thioester of a correctly functionalized hexaketide to form 1, but it is unable to initiate polyketide formation from malonyl-CoA. We show that the starter unit acyltransferase (SAT) of Hpm3 is critical for crosstalk between the two enzymes and that the rate of biosynthesis of 1 is determined by the rate of hexaketide formation by Hpm8.

Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Biosynthesis of lovastatin uses an iterative type I polyketide synthase (PKS). LovC is a trans-acting enoyl reductase (ER) that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. Such trans-acting ERs have been reported across a variety of other fungal PKS enzymes as a strategy in nature to diversify polyketides. How LovC achieves such specificity is unknown. The 1.9-Å structure of LovC reveals that LovC possesses a medium-chain dehydrogenase/reductase (MDR) fold with a unique monomeric assembly. Two LovC cocrystal structures and enzymological studies help elucidate the molecular basis of LovC specificity, define stereochemistry, and identify active-site residues. Sequence alignment indicates a general applicability to trans-acting ERs of fungal PKSs, as well as their potential application to directing biosynthesis.

Lovastatin, a cyclic nonaketide from Aspergillus terreus, is a hypercholesterolemic agent and a precursor to simvastatin, a semi-synthetic cholesterol-lowering drug. The biosynthesis of the lovastatin backbone (dihydromonacolin L) and the final 2-methylbutyryl decoration have been fully characterized. However, it remains unclear how two central reactions are catalyzed, namely, introduction of the 4a,5-double bond and hydroxylation at C-8. A cytochrome P450 gene, lovA, clustered with polyketide synthase lovB, has been a prime candidate for these reactions, but inability to obtain LovA recombinant enzyme has impeded detailed biochemical analyses. The synthetic codon optimization and/or N-terminal peptide replacement of lovA allowed the lovA expression in yeast (Saccharomyces cerevisiae). Both in vivo feeding and in vitro enzyme assays showed that LovA catalyzed the conversion of dihydromonacolin L acid to monacolin L acid and monacolin J acid, two proposed pathway intermediates in the biosynthesis of lovastatin. LovA was demonstrated to catalyze the regio- and stereo-specific hydroxylation of monacolin L acid to yield monacolin J acid. These results demonstrate that LovA is the single enzyme that performs both of the two elusive oxidative reactions in the lovastatin biosynthesis.