Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering

Abstract

DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

Combinatorial metabolic engineering platform enabling stable overproduction of lycopene from carbon dioxide by cyanobacteria

“…Firstly, we used a large-scale combinatorial approach to metabolic pathway construction. Such approaches have proven useful for simultaneously optimising expression of multiple genes in other organisms 37,38,45,46 , but have not been established in cyanobacteria. Only two small-scale examples of combinatorial designs have been reported, the first varying a small number of alternative enzyme-encoding sequences 60 and the second varying only the RBSs in a two-enzyme pathway 72 .…”

“…Several DNA assembly methods are suitable for combinatorial assembly 48 , but most are designed for a specific organism. Start-Stop Assembly 38 offers an ideal general-purpose DNA assembly system, with a unique streamlined assembly hierarchy which typically requires construction of only one new vector to apply the system to a new organism. Therefore we constructed a new Level 2 Start-Stop Assembly vector pGT270 ( Figure S1) for the cyanobacterium Synechocystis sp.…”

“…To test this platform, and to tackle genetic instability, overproduction of the terpenoid lycopene was adopted as a model system ( Figure 2). Lycopene is a valuable product used in the pharmaceutical, food and cosmetic industries 61 with a strong red colour which can be used as a measure of pathway activity 37,38,62 including by visual inspection of colony colour, at least in E. coli . Lycopene is produced endogenously by Synechocystis and other cyanobacteria as a precursor of other carotenoids used for photoprotection, but does not naturally accumulate ( Figure 2).…”

“…In the case of heterologous metabolic pathways, the diversion of central metabolites away from endogenous biosynthesis towards non-native products can limit growth. The relative expression of enzymes in heterologous pathways may also be poorly balanced, leading to accumulation of intermediates, which can be toxic 37,38 . Heterologous metabolic pathways which impair growth are genetically unstable, because selection will favour cells in which the pathways are inactivated by spontaneous point mutations or deletions, until such cells predominate.…”

“…The same assembly methods allow for optimisation by rational replacement of individual parts to fine-tune expression. This combinatorial approach to construction and optimisation of metabolic pathway-encoding constructs is a recent development, and while its effectiveness has been clearly demonstrated in model organisms like Escherichia coli and Saccharomyces cerevisae 37,38,45,46 , it is not yet standard practice, and in many other organisms including cyanobacteria it has not yet been established.…”