In Vitro Techniques for Studying PKSs

In order to fuUy understand the complex enzymatic processes carried out by PKS domains, in vitro studies are often carried out. Although in vivo studies are desirable, some of the organisms responsible for producing novel natural products are currently unculturable under laboratory conditions, placing the emphasis on in vitro studies to investigate such biosynthetic systems. 

The use of SNAC thioestes has many advantages. Firstly, the commercially available A-acetylcysteamine is far cheaper than CoA, making the synthesis of a range of substrate probes financially viable. In addition, the SNAC thioester offers reduced functionality, ranoving the potential side reactions during synthesis. Finally, the SNAC thioesters are far more stable than acyl-CoAs as labile phospho-ester bonds are no longer present. 

An improvement upon the SNAC thioester is likely to be an acyl-ACP, which are also used as substrate probes for PKS domains. The fundamental drawback to acyl-ACPs is that their synthesis is expensive and time consuming. The current methodology requires enzymatic transfer of the acyl-PPant group of an acyl-CoA to an apo-ACP using a phosphopantetheinyl transferase (PPTase) enzyme [96]. 

This approach suffers from several disadvantages. Firstly, it requires production of apo-kCP from a strain deficient in the 4 -phosphopantetheinyl transferase sfp gene. The subsequent synthesis of acyl-ACPs is often slow and requires either production or purchase of the PPTase enzyme. Furthermore, attachment of more elaborate acyl groups requires access to synthetically challenging and expensive acyl-CoAs. 

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