The Mechanism of Dehydrating Bimodules in trans‐Acyltransferase Polyketide Biosynthesis: A Showcase Study on Hepatoprotective Hangtaimycin

Abstract A bioassay‐guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis. The biosynthetic gene cluster was identified and resembles a hybrid trans‐AT PKS/NRPS biosynthetic machinery whose first PKS enzyme contains an internal dehydrating bimodule, which is usually found split in other trans‐AT PKSs. The mechanisms of such dehydrating bimodules have often been proposed, but have never been deeply investigated. Here we present in vivo mutations and in vitro enzymatic experiments that give first and detailed mechanistic insights into catalysis by dehydrating bimodules.

1 Supporting Information Table of Contents   Table S1. Bacterial Table S3. 13 Table S1. DNA manipulations were performed using standard procedures for E. coli and Streptomyces. The chemical reagents and antibiotics were purchased from Sigma-Aldrich. The test kits for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were purchased from Nanjing Jiancheng Bioengineering Institute. Oligonucleotide primers used in this study (Table S2) were synthesized by Tsingke. DNA sequencing of PCR products was recorded by Tsingke. Host for protein expression [2] pGro7 Host for protein expression Takara

CCTCC M2017417
Hangtaimycin (HTM) producing wild-type strain [3] ∆htm HTM biosynthetic gene cluster deletion mutant strain Figure S50 ∆htmA7 htmA7 in-frame deletion mutant strain Figure S51 DH1(H26A) Site-directed mutation strain of DH1 in module 1 of HtmA1 Figure S52 KR1(G10A, G12A, G15A) Site-directed mutation strain of KR1 in module 1 of HtmA1 Figure S53 KR2(Y168A) Site-directed mutation strain of KR2 in module 2 of HtmA1 Figure S55 DH3(H25A) Site-directed mutation strain of DH3 in module 3 of HtmA1 Figure S56 ACP2(S40A) Site-directed mutation strain of ACP2 in module 2 of HtmA1 Figure S57 ACP3(D43L, S44A) Site-directed mutation strain of ACP3 in module 3 of HtmA1 Figure S58 ∆KS3(C155A) Site-directed mutation strain of KS3 in module 3 of HtmA1 Figure S59 Plasmid pYH7 Streptomyces-E. coil shuttle vector [4] pET28a(+) Vector for protein expression Invitrogen pWHU5001 Recombinant plasmid used for HTM biosynthetic gene cluster deletion in vivo Figure S50 pWHU5002 Recombinant plasmid used for htmA7 in vivo in-frame deletion Figure S51 pWHU5003 Recombinant plasmid used for in vivo site-directed mutation of DH1 in module 1 of HtmA1 Figure S52 pWHU5004 Recombinant plasmid used for in vivo site-directed mutation of KR1 in module 1 of HtmA1 Figure S53 pWHU5005 Recombinant plasmid used for in vivo site-directed mutation of KR2 in module 2 of HtmA1 Figure S55 pWHU5006 Recombinant plasmid used for in vivo site-directed mutation of DH3 in module 3 of HtmA1 Figure S56 pWHU5007 Recombinant plasmid used for in vivo site-directed mutation of ACP2 in module 2 of HtmA1 Figure S57 pWHU5008 Recombinant plasmid used for in vivo site-directed mutation of ACP3 in module 3 of HtmA1 Figure S58 pWHU5009 Recombinant plasmid used for in vivo site-directed mutation of KS3 in module 3 of HtmA1 Figure Figure S59 pWHU5015 Recombinant plasmid used for HtmA7 (trans-ATs) protein expression in E. coli This work Table S2. List of oligonucleotide primers used in this study.

HtmM3-up GTGCCGCGCGGCAGCCATATGCACAGGGTCGCCGTGGTCGGC NdeI
HtmM3-re TGTCGACGGAGCTCGAATTCTCACGCAGGCTCGTGGTGGCGG Nucleotides in bold type are restriction sites introduced.

But-3-en-2-yl but-3-enoate (S12). TLC
To a solution of TFA / CH2Cl2 (2 : 3, 2.5 mL) was added S5 (106.9 mg, 0.44 mmol). The reaction was stirred at room temperature for 30 min and then concentrated under reduced pressure to give S6 as white solid which was used directly in the next step.

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Gene deletion or site-directed mutation in vivo. The recombinant plasmids used for gene disruption and site-directed mutation were introduced into S. spectabilis CCTCC M2017417 by conjugation using donor strain E. coli ET12567/pUZ8002 on ABB13 plates. After incubation at 28 °C for 12 h, the plate was overlaid with the final concentration of 35 µg/mL apramycin and 30 µg/mL nalidixic acid.
Exconjugants were selected on ABB13 plates supplied with 35 μg/mL apramycin and 30 μg/mL nalidixic acid to check their antibiotic resistance. Then single colonies were patched onto ABB13 plates containing 35 μg/mL apramycin and onto ABB13 plates without antibiotic, respectively, to screen for the double crossover mutant. The mutant candidates with correct phenotype (Apr R ) were further verified by PCR and sequencing with corresponding primers (Table S2).

Construction of gene deletion plasmids.
To knock out the htm gene cluster, two homologous recombination fragments of 2061 bp and 2060 bp flanking the ~92 kb htm cluster in the genome were amplified by PCR using two pairs of primers htm- L-up and htm-L-re, htm-R-up and htm-R-re, respectively (Table S2). These two fragments were then cloned into the Streptomyces-E. coli shuttle vectors pYH7 [4] treated with NdeI and HindIII by Gibson method to create the recombinant plasmid pWHU5001. To verify the plasmid and the mutant, a pair of primers htm-confirm-up and htm-confirm-re (Table S2) flanking the deletion region were used for PCR and sequencing. and ACP3, each two homologous recombination fragments were amplified by overlapping PCR using primer pairs listed in Table S2, then fused into the shuttle vector pYH7 digested with NdeI and HindIII to yield the corresponding recombinant plasmids (Table S1).

HtmA1
-  KR1 ACP1 ATd The changed nucleic acids and corresponding amino acid are shown in blue and marked with asterisks. A MluI restriction site which is used for mutant candidate screening by PCR is highlighted in yellow. The PCR product of mutation was confirmed by restriction enzyme digestion and sequencing. The plasmid pWHU5007 and wild-type genomic DNA were used as positive and negative control, respectively. coupling with another mutation from aspartic acid (D) to leucine (L). The changed nucleic acids and corresponding amino acids are shown in blue and marked with asterisks. A NheI restriction site which is used for mutant candidate screening by PCR is highlighted in yellow. The PCR product of mutation was confirmed by restriction enzyme digestion and sequencing. The plasmid pWHU5008 and wild-type genomic DNA were used as positive and negative control, respectively. transfer to ACP3 and dehydration. The expression plasmids for module 1, module 2, module 3 and module 3 containing   the mutated KS3 domain were generated by PCR amplifying with primes listed in Table S2, then inserted it into vector pET28a(+) to yield the corresponding recombinant plasmids (Table S1).

Synthesis of (E)-3-hydroxyhex-4-enoic acid (3)
To a solution of i Pr2NH (3.5 mL, 28 mmol) in THF (10 mL) was added n BuLi (10 mL, 2.5 M in hexane, 25 mmol) dropwise at -78 °C under N2. EtOAc (2 mL, 24 mmol) was then added dropwise. The resulting mixture was allowed to stir at -78 °C for 40 min, and then a solution of crotonaldehyde (1.6 mL, 20 mmol) in THF (2 mL) was added dropwise. After 1 h at -78 °C, the solution was poured into an ice-cold mixture of a saturated solution of NH4Cl and EtOAc. The resulting mixture was stirred vigorously for a few minutes, and the layers were separated. The aqueous layer was extracted with EtOAc twice. The combined organic layers were dried over MgSO4, filtered and concentrated to afford the ethyl (E)-3-hydroxyhex-4-enoate (rac)-S13 as a colorless oil. To the residue was added NaOH (3.2 g, 80 mmol), EtOH (14 mL) and H2O (4 mL). The resulting mixture was allowed to stir at 60 °C for 5 h. The solvent was removed under reduced pressure. The residue was dissolved in H2O, and then the aqueous solution was washed with Et2O. The aqueous solution was acidified to pH 1 with an aqueous solution of HCl (2 M) and extracted with EtOAc. The combined extracts were washed with brine, dried over Na2SO4, filtered and concentrated. Purification by column chromatography (5% MeOH in DCM) gave (E)- 3-hydroxyhex-4-enoic acid (3) [10] as a yellow solid (1.53 g, 67 % yield over 2 steps). 1