Module and individual domain deletions of NRPS to produce plipastatin derivatives in Bacillus subtilis

Abstract

Background: Plipastatin, an antifungal lipopeptide, is synthesized by a non-ribosomal peptide synthetase (NRPS) in Bacillus subtilis. However, little information is available on the combinatorial biosynthesis strategies applied in plipastatin biosynthetic pathway. In this study, we applied module or individual domain deletion strategies to engineer the plipastatin biosynthetic pathway, and investigated the effect of deletions on the plipastatin assembly line, as well as revealed the synthetic patterns of novel lipopeptides.

Results: Module deletion inactivated the entire enzyme complex, whereas individual domain (A/T domain) deletion within module 7 truncated the assembly line, resulting in truncated linear hexapeptides (C_16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val). Interestingly, within the module 6 catalytic unit, the effect of thiolation domain deletion differed from that of adenylation deletion. Absence of the T₆-domain resulted in a nonproductive strain, whereas deletion of the A₆-domain resulted in multiple assembly lines via module-skipping mechanism, generating three novel types of plipastatin derivatives, pentapeptides (C_16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu), hexapeptides (C_16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ile), and octapeptides (C_16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Gln-Tyr-Ile).

Conclusions: Notably, a unique module-skipping process occurred following deletion of the A₆-domain, which has not been previously reported for engineered NRPS systems. This finding provides new insight into the lipopeptides engineering. It is of significant importance for combinatorial approaches and should be taken into consideration in engineering non-ribosomal peptide biosynthetic pathways for generating novel lipopeptides.

Keywords: Genetic engineering; Module skipping; Novel lipopeptides; Plipastatin synthetase.

Fig. 1

a Schematic diagram of the plipastatin biosynthetic system. b Structure of plipastatin. c Deletion strategies: (i) deleting module 6 or 7 in plipastatin NRPS respectively, (ii) Deleting single A₆ or A₇ domain, (iii) deleting single T₆ and T₇ domain. Plipastatin NRPS assembly line contains five subunits, encoded by five genes ppsABCDE. Base on their function, the plipastatin synthetase are divided into 10 modules. Each module is comprised of condensation (C), adenylation (A) and thiolation (T) domains. Module 2, 4, 6 and 9 contains an epimerization (E) domian. A thioesterase (Te) domain is located on the C-teminus of module 10, which is responsible for products cyclisation and hydrolysis

Fig. 2

The high-resolution ESI-TOF–MS of hexapeptide ions with retention time (RT) 12.67–13.01 min produced by mutant strain BA7

Fig. 3

ESI-MS/MS spectra of protonated hexapeptide ions [M + H]⁺ at m/z 980.5579 a and m/z 1008.5878 b, acquired in Quadrupole-TOF (Q-TOF) mass spectrometer of crude extract from mutant strain BA7

Fig. 4

a LC–ESI–MS total chromatogram of crude extract from mutant strain BA6. b Chromatogram corresponding to m/z 1327.7401. c Chromatogram corresponding to m/z 923.5341. d Chromatogram corresponding to m/z 1036.6182. e High-resolution ESI–MS of parent ions from compound 1, 2 and 3

Fig. 5

MS/MS analysis of octapeptide produced by mutant strain BA6. a The sequence of octapeptide and thirteen characteristic product ions b₁ ~ b₇ and y₂ ~ y₇. b MS/MS spectrum of octapeptide ion [M + H]⁺ at m/z 1327.7378 and assignment of key product ions

Fig. 6

MS/MS analysis of pentapeptide produced by mutant strain BA6. a The sequence of pentapeptide and seven characteristic product ions b₁ ~ b₄ and y₂ ~ y₄. b MS/MS spectrum of pentapeptide ion [M + H]⁺ at m/z 923.5330 and assignment of key product ions

Fig. 7

MS/MS analysis of hexapeptide produced by mutant strain BA6. a The sequence of hexapeptide and nine characteristic product ions b₁ ~ b₅ and y₂ ~ y₅. b MS/MS spectrum of hexapeptide ion [M + H]⁺ at m/z 1036.6172 and assignment of key product ions

Fig. 8

An assumed mechanism involving module skipping for mutant plipastatin NRPS without A₆ domain. a Formation of linear octapeptide by complete module 6 and 7 skipping. b Formation of linear hexapeptide by complete module 6, 7, 8 and 9 skipping. c Formation of linear pentapeptide by hydrolysis in advance of thioesterase (Te) domain