In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Abstract

Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide biosynthesis, and provides a tunable platform with advantages over conventional methods for protein expression. Here, we demonstrate the use of cell-free protein synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC–MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first step to apply cell-free protein synthesis to discover and characterize new natural products.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…The development of a S. venezuelae TX-TL system potentially provides a fast route to obtaining enzymes from Streptomyces secondary metabolism using a homologous host for protein folding. Whilst each protein target is unique, we will investigate this tool for the synthesis of specialized enzymes that require post-translational modification [10,11] or exotic precursors for protein folding, such as coenzyme F 420 [12]. We will now focus on enhancing this initial S. venezuelae TX-TL platform, prototype gene circuits and investigate its use for the characterization of cryptic gene clusters located within the Actinomycetes bacteria [23,24] .…”

“…One potential new application for TX-TL is the direct assembly of natural products from biosynthetic genes, as recently pioneered in E. coli TX-TL for the co-synthesis of two large (> 100 kDa) non-ribosomal peptide synthetases [10]. Indeed, for expression of genes from Streptomyces species, E. coli may not be the ideal host chassis in all cases -e.g.…”

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

“…Because of the absence of cell walls, the reaction environment is open, accessible, and controllable, allowing for direct and easy manipulation, monitoring, sampling, and optimization. CFPS has also been applied for the rapid prototyping of biological circuits and metabolic pathways (Garamella et al, 2016;Karim and Jewett, 2016;Sun et al, 2014;Takahashi et al, 2015), biosynthesis of natural products (Goering et al, 2017) as well as designing of paperbased diagnostics (Pardee et al, 2014(Pardee et al, , 2016a. CFPS systems help address a growing need for simple, inexpensive, and efficient protein production technologies.…”

“…So far, they have been widely utilized for manufacturing a wide variety of active protein products that include therapeutic vaccines (Kanter et al, 2007;Yang et al, 2005), antibodies (Min et al, 2016;Stech and Kubick, 2015), viruslike particles (Bundy et al, 2008;Lu et al, 2015), membrane proteins (Henrich et al, 2015;Sachse et al, 2014), metalloproteins (Boyer et al, 2008;Kwon et al, 2013;Li et al, 2016) and proteins harboring non-standard amino acids (Hong et al, 2014(Hong et al, , 2015. CFPS has also been applied for the rapid prototyping of biological circuits and metabolic pathways (Garamella et al, 2016;Karim and Jewett, 2016;Sun et al, 2014;Takahashi et al, 2015), biosynthesis of natural products (Goering et al, 2017) as well as designing of paperbased diagnostics (Pardee et al, 2014(Pardee et al, , 2016a. The ability to freezedry CFPS systems is opening the way to novel on demand biomanufacturing applications as well (Dudley et al, 2016;Pardee et al, 2016b;Salehi et al, 2016;Smith et al, 2014).…”

In vitroprototyping and rapid optimization of biosynthetic enzymes for cellular design

“…[27][28][29] For instance, the Panke group has shown that DHAP can be made in crude lysates and real-time monitoring can optimize production. 29 In addition, our group has shown that 2,3-butanediol, 30 mevalonate, 28 n-butanol, 27,31 limonene, 32,33 and more complex products 34 can be constructed in crude lysates with high productivities (>g/L/h). However, to our knowledge, no attempts have been made using cell-free prototyping to improve engineering of industrially-relevant, non-model organisms.…”