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. 2021 Sep 10;87(19):e0106621.
doi: 10.1128/AEM.01066-21. Epub 2021 Sep 10.

Enhancing Ristomycin A Production by Overexpression of ParB-Like StrR Family Regulators Controlling the Biosynthesis Genes

Kai Liu  1 , Xin-Rui Hu  1 , Li-Xing Zhao  2 , Yemin Wang  1 , Zixin Deng  1 , Meifeng Tao  1
Affiliations

Enhancing Ristomycin A Production by Overexpression of ParB-Like StrR Family Regulators Controlling the Biosynthesis Genes

Kai Liu et al. Appl Environ Microbiol. .

Abstract

Amycolatopsis sp. strain TNS106 harbors a ristomycin-biosynthetic gene cluster (asr) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene, asrR, abolished ristomycin A production and the transcription of the asr genes orf5 to orf39. The ristomycin A fermentation titer in Amycolatopsis sp. strain TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene, bbr, from the balhimycin-biosynthetic gene cluster (BGC) utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in a fermentation titer of 4.01 g/liter in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR operon and probably orf12 to orf14 but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr. IMPORTANCE This study showed that overexpression of either a native StrR family regulator (AsrR) or a heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production by increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotic biosynthesis. Furthermore, the regulator overexpression plasmids constructed in this study could serve as valuable tools for strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effects. The overproduction strains engineered in this study are ideal materials for industrial production of ristomycin A.

Keywords: Amycolatopsis; N-terminal ParB-like domain; StrR family regulator; antibiotic titer improvement; glycopeptide antibiotics; ristomycin.

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Figures

FIG 1
FIG 1
Identification of ristomycin A produced by Amycolatopsis sp. strain TNS106. (A) Chemical structure of ristomycin A. (B) HPLC profile analysis of the ristomycin A standard and fermentation broth supernatant of strain Amycolatopsis sp. strain TNS106 (WT) cultured in SAM medium for 2 days. (C and D) UPLC-HRESI-MS analysis (positive model) of the ristomycin A standard and sample collected from the extract of WT fermentation. The molecular ion peak [M+2H]2+ was observed at m/z =1,034.3392 for the standard and m/z = 1,034.3398 for the sample.
FIG 2
FIG 2
The cluster-situated regulator AsrR is indispensable for the biosynthesis of ristomycin A. (A) HPLC profile analysis of ristomycin A in WT, ΔasrR, ΔasrR::pJLK89 (ΔasrR complemented with asrR under the control of kasOp*), and ΔasrR::pJLK91 (ΔasrR complemented with bbr under the control of kasOp*) strains. The ΔasrR::pJLK83 (ΔasrR with empty vector) mutant was used as the control strain. All strains were cultured in SAM medium for 7 days, and samples for HPLC qualitative analysis were collected on days 1 to 7 from fermentation cultures. The HPLC chromatogram shows results for samples of strains collected on day 2. (B) Quantitative analysis of ristomycin A in WT, ΔasrR, ΔasrR::pJLK83, ΔasrR::pJLK89, and ΔasrR::pJLK91 strains. Samples for quantitative analysis were collected on the second day from fermentation cultures. Data are from three independent biological replicates of each strain.
FIG 3
FIG 3
Exploring the genetic manipulation levels of asrR and bbr to assess the impact on ristomycin A titer evaluated by HPLC. WT strains with empty vectors pIB139 and pJLK83 were designated control strains WT::pIB139 and WT::pJLK83, respectively. WeA and WeB are WT with asrR and bbr, respectively, under the control of the promoter ermEp*. WkA and WkB are WT with asrR and bbr, respectively, under the control of the promoter kasOp*. WAB is WT with asrR and bbr expressed tandemly under the control of kasOp*. W2B and W3B are WT with two and three copies, respectively, of bbr under the control of kasOp*. All strains were cultured in SAM medium for 7 days, and samples for HPLC qualitative analysis were collected on days 1 to 7 from fermentation cultures. The HPLC chromatogram shows analysis results for samples of strains collected on day 2.
FIG 4
FIG 4
Quantitative analysis of ristomycin A, dry biomass, and asr gene transcription in the asrR and bbr overexpression strains. (A and B) Quantitative analysis of ristomycin A. (C and D) Quantitative analysis of the dry weight of mycelium. (E and F) RT-qPCR analysis of asr gene transcription. Control strains included the WT and the WT with the empty vectors pIB139 (WT::pIB139) and pJLK83 (WT::pJLK83). See the text or Fig. 3 for information about the engineered strains WeA to W3B. The genes vanX (orf3), rpsB (orf8), mtfB (orf23), pgat (orf24), gtfF (orf34), and dahp (orf39) were selected to examine expression of asr. Results are shown for three independent biological replicates of each strain. Error bars show standard deviations (SD). *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Ristomycin-biosynthetic gene cluster and analysis of transcriptional organization. (A) Organization of the ristomycin-biosynthetic gene cluster in Amycolatopsis sp. strain TNS106, including 39 ORFs (Table 1). The horizontal arrows show the transcriptional units and their orientation. The inverted triangles show the putative promoter regions (P-I, P-II, P-III, P-IV, P-V, P-VI, and P-VII) of the transcriptional units. The sizes of the intergenic regions (in bp) are shown beneath the genes and are marked by narrow, vertical gray arrows. (B) RT-PCR analysis of the asr from Amycolatopsis sp. strain TNS106. The cDNA was generated by reverse transcription of total RNA samples, genomic DNA and total RNA were used as control. An asterisk indicates that the neighboring genes were not part of the same transcriptional unit.
FIG 6
FIG 6
The P-II promoter region and binding sites recognized by AsrR or its homolog Bbr. (A) DNase I footprinting analysis of AsrR with probe dP-II. A 39-nt sequence containing 7-bp IRs (underlined nucleotides) separated by a 15-bp internal spacer was protected by AsrR. (B) Sequence of the P-II promoter region (operon comprising orf4 and orf5) and binding site of AsrR. Boldface indicates the asrR transcription start site (TSS). Boxes, putative −10 and −35 regions and start codon; gray shading, AsrR binding site. (C) The probes P-ii, P-iv, P-vi, P-vii, and P-12* were derived from the putative promoter regions P-II, P-IV, P-VI, and P-VII and the region upstream of orf12 (probe P-12), respectively, based on in silico analysis. Boxes, IRs; dashed box, protected sequence (39 nt); red font, same nucleotide as in the P-II IRs. (D) Mutagenized versions of probes P-ii, P-iv, P-vi, and P-vii. Boxes, IRs; gray shading, mutagenized bases; red font, same nucleotide as in the P-II IRs. (E and F) EMSAs with (E) AsrR or (F) Bbr and the P-ii probe or mutagenized probes m1P-ii and m2P-ii. (G and H) EMSAs with (G) AsrR or (H) Bbr and probes mP-iv, mP-vi, mP-vii, and P-12*.
FIG 7
FIG 7
Function analysis of the conserved sites within the N-terminal ParB-like domain of AsrR and Bbr. (A) Amino acid sequence alignment of the ParB-like domains of AsrR, Bbr, and StrR and the N-terminal domains of well-studied ParB homologues PadC_Mx (from Myxococcus xanthus; UniProt accession number Q1D3H3) and ParB_Bs (from Bacillus subtilis; UniProt accession numbers P26497). Five residues mutated in AsrR or Bbr are indicated by vertical arrows. The conserved residues are in red. High amino acid similarity values (>50%) are indicated by blue font. Gray shading indicates residues that mediate the interaction of PadC_Mx with CTP (32) and ParB_Bs with CDP (33). The conserved GxxRxxA motif in SFRs and ParB homologues is marked by a dashed box. The schematic below the alignment shows the secondary structure of ParB_Bs, based on its crystal structure (PDB database accession number 6SDK) (33). (B) Activation of ristomycin A production by AsrR and its point mutants in ΔasrR. Integrative plasmids carrying wild-type asrR and point mutants of asrR and bbr under the control of kasOp* were integrated into the chromosome of ΔasrR individually. The resultant strains were fermented, and ristomycin A was quantitatively analyzed by HPLC. Data are from three independent biological replicates. Error bars, SD. *, P < 0.05. (C) Analysis of DNA affinity of AsrRm5A and Bbrm5A with probe P-ii by EMSA.
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