Cervecería Nacional (Cerveceria Nacional, 2016)

biosynthesis

In natural product research, mass spectrometry is a key analytical technique. The rapid identification of both small and large molecules, can be particularly advantageous for high throughput screening. Highly sensitive instruments allow detection of compounds from both low sample volumes and low concentrations, as well as from complex samples. Structural elucidation of compounds can be begun by MS; however, NMR would still be the technique of choice for structural characterisation.

1.6.5.1. The use of isotope labelling in mass spectrometry

Often, labelled precursors, such as 13C sodium acetate, are fed to organisms, and their uptake by PKSs is monitored by MS. High resolution MS instruments can identify molecular formulae of products to a high degree of accuracy, for example, sub ppm accuracy on a FTICR-MS is routine, and with detailed calibration, sub ppb is possible. Detecting mass differences caused by 13C or deuterium labelling is simple for most modern instruments.

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1.6.5.2. Fragmentation techniques for natural product research

CAD is the most utilised technique for structural characterisation of small molecules and proteins. However, newer modes of fragmentation, such as EID and ECD, can provide structural information where CAD fails. Electron based fragmentation techniques can produce different fragments to those seen with CAD. Håkansson and coworkers have shown that it is possible to determine the location of double bonds in fatty acids by EID, combined with manganese adduction190, and in the O’Connor group EID was able to differentiate between two isomers, lasalocid A 1 and iso- lasalocid A.191

1.6.5.2.1. Fragmentation of the phosphopantetheinyl (PPant) arm of carrier proteins

CAD has proven especially useful in the identification and characterisation of ACPs, and in generating characteristic PPant ejection ions 58. Upon CAD fragmentation, the phosphodiester bond adjacent to the pantetheinyl moiety is cleaved providing 58, or alternatively the phosphodiester bond adjacent to the protein is broken, generating the larger phospho-Pant ejection ion 59. This technique has been used in proteomics for identifying PKSs, but also in off-loading intermediates from ACP-bound species.157-159, 192, 193 IRMPD also provides this Pant ion but fewer instruments are equipped with this tool for fragmentation (Scheme 13).

A drawback to the “PPant ejection assay” is that it requires a degree of skill by the MS user.157 The assay has, however, been used effectively on several occasions.141,

142, 192, 194

In particular, intermediates from the lovastatin 5 biosynthetic pathway, a non-iterative type I PKS, were trapped by performing a trypsin digest of the 277 kDa

60 megasynthase once the PKS had been activated in vitro, rendering it inactive. The resulting peptides were fragmented, and intermediates, bound to the ejected Pant ion

58/59, were detected, with additional 13C labelling for confirmation.192 Similarly, an

enzyme bound intermediate was detected with the “PPant ejection assay” from the enediyne C-1027 pathway, and the result aided the understanding of the biosynthetic pathway.195

Scheme 12 Generation of a pantetheine (Pant) ejection ion 58 or a phospho-Pant ejection ion 59 from the corresponding ACPby CAD or IRMPD.

1.6.5.2.2. Proteomics based approaches to detect and characterise PKSs

Bafna and co-workers developed a proteomics based method that combines enriching the abundance of PKSs in the MS sample by affinity tagging TE domains, followed by tryptic digestion, and then identification of PPant active sites either by MS3 or by machine learning from the MS2 spectra. The machine-learning approach allows lower cost instruments to use a proteomics approach to identify PKSs without MS3, which is often out of reach for those instruments.157

A similar method from the Burkart laboratory, called the Orthogonal Active Site Identification System (OASIS), uses active site probes to enrich the carrier protein and thioesterase peptides in complex biological samples. The carrier proteins are

61 labelled either by incorporation of CoA precursors with azide groups for chemoselective ligation to reporters with alkyne groups or with biotinylated CoA analogues for purification with streptavidin. Thioesterases can be enriched by modification of the active site serine using fluorophosphonate with a biotin linker for pull down. The samples are then analysed by MudPIT196, 197 (Multidimensional Protein Identification Technology), which is a 2D LC-MS technology for separating and identifying individual peptides and proteins from complex samples. PKSs and NRPSs are identified by cross-referencing the peptides identified by each probe. Thus far, OASIS has only been demonstrated on a model bacteria, B. subtilis, where they successfully detected and identified all of the modular synthases expressed in that organism.160

Proteomics-based approaches to discovering PKSs have also proven successful in the Kelleher group, combined with the “PPant ejection assay” to find ACPs and identify the PKS from that information. One such platform is the Proteomic Investigation of Secondary Metabolism (PrISM).159 In this way, only the expressed NRPS/PKSs are detected, avoiding traditional genome mining methods that cannot distinguish between expressed or ‘silent’ gene clusters. Cultures of the bacteria are first screened for the presence of high molecular weight proteins, as those proteins are likely to be NRPSs or PKSs. The bacteria are grown in different conditions, the large proteins (150 kDa or more) are analysed by MS and their peptide fragments from digestion are used to identify the biosynthetic machinery.

Once the carrier protein has been found and its sequence deduced it is possible to design primers to pull down the gene and then identify the full PKS gene, even if the genome sequence of the organism is not known. With that information, it is then possible to search for the natural product produced. Koranimine, a cyclic imine198,

62 flavopeptins, a class of peptide aldehydes,199 and gobichelins A and B, siderophores produced by NRPSs,200 were detected for the first time using PrISM.