Engineering of FK520 polyketide synthase for rapid access to quality control reference standards

Despite the ability of mega synthases to construct complex structures with high precision, the main product is often accompanied by minor congeners because of relaxed selectivity of biosynthetic steps. In the case of polyketides, these may arise from promiscuity of acyltransferase domains while selecting the extender building blocks. This yields compounds which can differ only slightly from the main product and are therefore notoriously difficult to separate. For compounds in medicinal use, such congeners are considered impurities and must be precisely quantified in commercial production through use of reference standards. The immunosuppressants FK506, FK520, and its chlorinated derivate pimecrolimus, are well known for accumulation of minor congeners in the fermentation, which demands costly and lengthy purification with industrial HPLC to obtain material of sufficient purity. The relaxed selectivity of the AT domain in module 4 of FK506/520 PKS results in production of FK506, FK506D, FK520, and FK523 from the same system. Here, we investigate additional minor impurities, specifically the 11-, 17-, and 19-ethyl FK520 derivatives. Because the PKS can process these analogues to completion, it is reasonable to expect that strategic replacement of AT domains in modules 9, 6, and 5 (respectively), would enhance their production. Indeed, we show a direct, exclusive and high titer biosynthesis of these analogues through PKS engineering, replacing tedious and costly isolation by preparative HPLC and significantly improving access to these minor congeners. In addition, by using the AT4 as the donor domain, an interesting system is obtained, containing identical promiscuous AT domains at two different positions, and consequently distinct intra- and intermodular context. Media-dependent extender pool modulation toward methylmalonyl-CoA showed unexpected but consistent distribution of the four predicted analogues for each of the engineered systems. Despite the use of identical ATs, extender incorporation preference is different, depending on the modular context. It appears that the selectivity of the downstream AT4-containing module depends on the structural features of the incoming acyl chain; features installed by a module well upstream in the assembly line. Therefore, the global context of the PKS may be more impactful to the outcome of AT-engineering experiments than is generally considered.