Abstract
Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomyces tsukubaensis YN06, we found that AT4FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.
Highlights
The resulting S. tsukubaensis strains YN06-01 (PAC-B18), YN06-02 (PAC-B18-V187K) and wild type were grown on ISP4 plates for 5–9 days at 28◦C to allow colonies to sporulate for fermentation
We have reported that AT4FkbB in FK506 polyketide synthase (PKS) can transfer both allmal and ethmal units to ACP4FkbB, accounting for simultaneous production of FK506 and FK520 in S. tsukubaensis YN06 (Jiang et al, 2015)
HPLC data showed that a new large peak appeared along with AT4FkbB (Figures 1A–D), and this HPLC peak contained two products, which were identical to the biochemically synthesized allmal- and ethmal-AT4FkbB based on mass spectrum (MS) data, indicating self -acylation has occurred on AT4FkbB
Summary
Polyketide natural products function as a wide range of therapeutic agents, such as immunosuppressants (Mo et al, 2011), anti-cancer agents (Dunn et al, 2013), antibiotics (Urem et al, 2016), which are invaluable natural resources for pharmaceutical development (Fegan et al, 2010; Dunn et al, 2013; Mo and Suh, 2016). The essential catalytic module is basically comprised of β-ketoacyl synthase domain (KS), AT, and ACP domain (Fischbach and Walsh, 2006; Khosla et al, 2007; Fegan et al, 2010; Dunn et al, 2013). The method of dipparental mating was accoding to Jones et al (2013) to obtain the ET12567 strains containing PAC-B18 or PACB18-V187K (ApraR) and pUZ8002 (KanR). The culture condition was maintained at 28◦C and 220 rpm for 5 days (Wang et al, 2016). Culture samples (2 ml) from each S. tsukubaensis strain were withdrawn and ultrasonic extracted with 500 μl of methanol. The FK506 and FK520 titers were calculated with standard curves
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
