Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like β-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching-acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

β-Lactone natural products occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from β-hydroxy-α-amino acids, which are themselves valuable chiral building blocks for chemical synthesis and precursors to numerous important medicines. However, despite a number of excellent synthetic methods for their asymmetric synthesis, few effective enzymatic tools exist for their preparation. Here we report cloning of the biosynthetic gene cluster for the β-lactone antibiotic obafluorin and delineate its biosynthetic pathway. We identify a nonribosomal peptide synthetase with an unusual domain architecture and an L-threonine:4-nitrophenylacetaldehyde transaldolase responsible for (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate biosynthesis. Phylogenetic analysis sheds light on the evolutionary origin of this rare enzyme family and identifies further gene clusters encoding L-threonine transaldolases. We also present preliminary data suggesting that L-threonine transaldolases might be useful for the preparation of L-threo-β-hydroxy-α-amino acids.

Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, 13 C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPHdependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.