Diversity oriented biosynthesis via accelerated evolution of modular gene clusters

Abstract

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.

A pharmaceutical model for the molecular evolution of microbial natural products

“…A similar approach resulted in the successful engineering of the rapamycin (Fig. C), biosynthetic pathway, which is a type I polyketide, biosynthetic pathway . Serendipitous natural recombination events were used to generate a library of novel analogs directly in a rapamycin‐producing Streptomyces strain .…”

“…C), biosynthetic pathway, which is a type I polyketide, biosynthetic pathway . Serendipitous natural recombination events were used to generate a library of novel analogs directly in a rapamycin‐producing Streptomyces strain . A temperature‐sensitive plasmid was first integrated into the gene cluster using homologous recombination.…”

“…A temperature‐sensitive plasmid was first integrated into the gene cluster using homologous recombination. Surprisingly, activation of the temperature‐sensitive replicon, while still integrated into the genome, led to the production of twenty chemical variants of rapamycin . Genome sequencing revealed that the progeny harbored new PKS architectures, including both deletion and duplication of modules, which were generated at positions of high‐sequence similarity .…”

De novo Biosynthesis of “Non‐Natural” Thaxtomin Phytotoxins

“…Many valuable therapeutic agents and agrochemicals are derived from natural products.H owever,m any of these compounds do not have the necessary bioactivity or physicochemical properties at the outset, and further derivatization or analogue synthesis is required to develop optimized, effective compounds.T his optimization is traditionally achieved through semi-synthesis or total synthesis.H owever, multistep chemical synthesis often requires deleterious reagents and can be costly,w hich is particularly problematic for the development of agrochemicals that need to be produced in large quantities.B iosynthetic engineering is an alternative approach to produce new and potentially improved natural product variants directly by fermentation. [1,2] Although biosynthetic engineering has been used to generate novel "non-natural" products [1][2][3][4][5][6] most approaches have been limited to manipulating biosynthetic pathways in the well characterized native producers.H owever there are many microorganisms that produce useful secondary metab-olites,which are not amenable to such genetic manipulation. Ther ise of synthetic biology has provided access to larger synthetic DNAc onstructs,r apid DNAc apture, [7][8][9] editing, [10,11] assembly, [12,13] and other advances.…”

“…In this paper we describe anew approach for the de novo biosynthesis of "non-natural" variants of thaxtomin phytotoxins produced by Streptomyces scabies ( Figure 1A), which inhibit cellulose biosynthesis in plants. [18,19] Thaxtomins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)are biosynthesized from l-phenylalanine and the unusual l-4-nitrotryptophan, which is produced by TxtD and TxtE enzymes ( Figure 1B). [20][21][22][23] TxtD is an itric oxide (NO) synthase and TxtE, aP 450 enzyme,t hen utilizes this NO with O 2 ,t oc atalyze nitration at the 4-position of l-tryptophan.…”