Novel biosynthesis pathway for aniline derivatives
Shunsuke MASUO
Faculty of Life and Environmental Sciences, University of Tsukuba
Research aims
Aniline derivatives are important compounds that can be used as raw materials forpharmaceuticals and chemicals. Recently, we discovered a novel pyrazine biosynthesis genecluster in Pseudomonas fluorescens.1) This gene cluster contains the 4-aminophenylalanine(4APhe) biosynthesis gene papABC and the pyrazine-ring biosynthesis gene papDEF, whichis involved in the production of 4-aminobenzyl-substituted pyrazines. Using the genome
mining approach, we discovered a gene cluster containing a putative non-ribosomal peptide
synthase (NRPS) gene and papABC-like genes in Haliangium ochraceum (Fig. 1A). Here,
we tried to understand the function of this gene cluster and to reveal the biosynthesis
mechanism of novel aniline derivatives.
Methods
1. Plasmid construction
Gene fragments were amplified by PCR using KOD One (Toyobo), appropriate primers, andthe genomic DNA of H. ochraceum JCM 11303, and were cloned into expression vectors
using the NEB Builder HiFi DNA Assembly (NEB). Sequences were confirmed bynext-generation sequencing and Sanger sequencing.
2. LC-MS analysis
The E. coli culture supernatant and ethyl acetate extraction fractions were subjected toLC-MS analysis using LCMS 8045 (Shimadzu). Metabolites were separated using anACQUITY UPLC BEH Shield RP18 Column (Waters).
Results
1. Domain prediction of the novel non-ribosomal peptide synthaseIn silico analysis using AntiSmash 5.1
predicted that Hoch1367 codes an NRPS
consisting of five modules (Fig. 1B). Basedon comparison of the amino acid residues
involved in substrate recognition that wereconserved in the adenylation (A) domain,
modules 1, 2, 4, and 5 were predicted toA

Haliangium ochraceum JCM11303

Hoch_1366_1367_1368 _1369 _1370_1371_1372

Hoch_1366…PapA-like; _1367…NRPS; _1368…PapC-like_1369…Methyltransferase; _1370…Oxidase_1371…PapB-like; _1372…Dioxygenase

B

Hoch_1367
Module 1 Module 2 Module 3 Module 4 Module 5
Cal T

C

A T C

A TC

A T C

A T C

A T

Figure 2．Gene cluster(A)and NRPS (B)recognize serine, serine, proline, and glycine, respectively. The N-terminal acyl-CoA ligase(CAL) domain could be involved in the acylation of serine in module 1.2) On the contrary,none of the residues in module 3 showed high homology with known substrate recognition
sequences, suggesting that module 3 recognizes a previously unknown substrate. As
Hoch_1367 clusters with putative 4-aminophenylalanine (4APhe) biosynthesis genes, it isassumed that module 3 recognizes 4APhe or its derivatives.
2. Hoch_1366, _1368, and _1370 encode PapA, PapB, and PapC, respectively to produce
4APhe
Hoch_1366, Hoch_1368, and Hoch_1370 were heterologously expressed in Escherichia coli
BL21 (DE3). HPLC analysis showed that E. coli expressing Hoch_1366, _1368, and _1370
accumulated 4APhe in the culture supernatant, whereas E. coli expressing only Hoch_1366
or Hoch_1368 and 1370 did not. These results indicated that Hoch_1366, Hoch_1368, and
Hoch_1370 function as PapA, PapB, and PapC, respectively, to produce 4APhe.
3. Functional analysis of the novel genecluster
To express active NRPS, we generated E.
coliexpressing
phosphopantetheinyltransferase (PPTase), which is involved in
the post-translational modification of NRPS.
The mtaA gene, which encodes PPTase withbroad substrate specificity derived from themyxobacteria Stigmatella aurantiaca, wasA

pET-HochOp (34 kb)

B

Hoch_1367

pCOLA-1367 (23 kb)

fused with the T7lac promoter. This fragment
was then introduced into the entD locus of E.
coli BL21 (DE3), HMS (DE3), and BL21
Star (DE3) to generate NRPS expression

pET-1363-75 (23 kb)
T7lac promoter

T7 terminatorhosts BLmtaA, HMSmtaA, and BLSmtaA,
Figure 2．Expression plasmids constructed inrespectively. A 27 kb DNA fragment
containing cluster genes from Hoch_1366 to this study.
Hoch_1372 was cloned into the T7lac
promoter-driven expression plasmids (Fig. 2A), and introduced into each NRPS expression
host. The resulting strains were then cultured in various media, temperatures, and IPTG
concentrations, and metabolites after IPTG induction, and then subjected to LC-MS analysis.
Unfortunately, no specific metabolites were identified in the strain harboring the expression
plasmid compared to the control strains harboring the empty plasmid. We constructed
another expression plasmid, in which each cluster gene was independently cloned under theT7lac promoter (Fig. 2B) and introduced into the NRPS expression host. We are currently
performing LC-MS analysis to detect the metabolites produced by the heterologously
expressed gene cluster from H. ochraceum.
4. Substrate recognition by the A domain of module 3
To analyze the function of module 3, which consists of the condensation (C), A, and PCP (T)domains (Fig. 2B), we attempted to prepare the recombinant N-terminal or C-terminal
His-tagged module 3. Although the target protein was expressed as inclusion bodies in mostE. coli hosts and culture conditions, small amounts of soluble expression were observed
when cultured at low temperatures in ArcticExpress (DE3) RP. Recombinant module 3 wasthen prepared by affinity purification using Ni-NTA beads. We will next measure theadenylation activities of module 3 against 4APhe and its derivatives.
Conclusion
In this study, we discovered a novel gene cluster containing the NRPS-like protein that is
considered to recognize 4APhe derivatives. Hoch_1366, _1368, and _1370 in the cluster
encode PapA, PapB, and PapC, respectively, and were identified as 4APhe biosynthetic
genes. This research focused on heterologous expression in E. coli, but in future, we would
like to utilize other expression hosts such as actinomycetes and myxobacteria. Further, wewould like to construct gene deletion strains of H. ochraceum. Our study will thus reveal thefunctions of novel NRPS and related enzymes and identify novel aniline-structure-containing
peptide compounds.
References
1) Masuo, S., Tsuda, Y., Namai, T., Minakawa, H., Shigemoto, R., and Takaya, N. (2020)
Enzymatic Cascade in Pseudomonas that Produces Pyrazine from α-amino acids.
ChemBioChem 21: 353-359.
2) Baars, O., Zhang, X., Gibson, M.I., Stone, A.T., Morel, F.M.M., and Seyedsayamdost,
M.R. (2018) Crochelins: Siderophores with an Unprecedented Iron-Chelating Moiety
from the Nitrogen-Fixing Bacterium Azotobacter chroococcum. Angew Chem Int Ed Engl.
57: 536-541.