Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering

Abstract

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified. • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Keywords: Apramycin; Biosynthesis; Carbamoyltobramycin; Lrp/AsnC; Oxygenase; Transcriptional regulator.

Fig. 1

Putative biosynthetic pathways of apramycin, tobramycin, and kanamycin B. The structures highlighted are those undergoing one or more enzymatic catalytic processes

Fig. 2

Elimination of apramycin in S. tenebrarius. a HPLC chromatogram of Tb and mutants. The blue curve represents the peak of apramycin, while the red curve represents the peak of carbamoyltobramycin. b Fermentation products of Tb and mutants analyzed by HPLC. Mean values of 3 replicates are shown, with the standards indicated by error bars

Fig. 3

Structural analysis of TobR protein. The structure of TobR was predicted by Alphafold 2, while other protein structures belongs to Lrp/AsnC family have been reported in the PDB database (PDB ID: 1i1g, 2dbb, 2ia0, 2yx4, 2cfx, 2e1c, 2p6s, 4pcq, 2cg4, 2gqq, and 2vby)

Fig. 4

The impact of TobR on S. tenebrarius. a Accumulation of biomass in liquid culture of Tb-△aprJ and Tb-△aprJ-△tobR. b Growth state of Tb-△aprJ and Tb-△aprJ-△tobR in MS solid medium. c Carbamoyltobramycin production of tobR disruption and overexpressed strains analyzed by HPLC. Mean values of 3 replicates are shown, with the standards indicated by error bars

Fig. 5

Changes in transcription levels of related genes after knocking out tobR at 72 h in fermentation

Fig. 6

EMSA Analysis of TobR and P_tobO. a SDS-PAGE of TobR. M, protein ladder; (1) whole cell; (2) supernatant; (3) precipitation; (4) crude protein/supernatant; (5) pure protein/TobR. b EMSA analysis of TobR binding to P_tobO, (a) P_tobO probe; (b) TobR-P_tobO complex. c Transcription unit of tobramycin biosynthetic gene cluster. P_tobE, P_tobT, P_tobB, P_tobZ, P_tobS1, P_tobM1, P_tobA, and P_tobO represented probes of promoter regions of each transcription unit. The genes highlighted in green in the cluster were speculated to be related to the tobramycin biosynthesis pathway. The genes highlighted in blue were speculated to encode transport proteins. The white markers represent proteins with unknown functions in tobramycin biosynthesis, and the red markers represent potential transcriptional regulatory factors. d The regulatory targets of TobR on tobramycin biosynthesis gene cluster analyzed by EMSA

Fig. 7

Bioinformatics analysis of TobO. a The amino acid sequences of TobO and its homologous proteins (blue background indicating the protein percent identity, the red box labeled amino acid represents the catalytic triad formed with ferrous ion; asparagine oxygenase PDB ID: 2OG5 from Streptomyces coelicolor has a protein similarity of 47.0% with TobO). b The structure of TobO predicted by the online tool Αlphafold 2. c Catalytic reaction of L-asparagine hydroxylase

Fig. 8

The impact of TobO on S. tenebrarius. a Carbamoyltobramycin production of tobO disruption and overexpressed strains by HPLC analysis. b Accumulation of biomass in liquid culture of Tb-△aprJ and Tb-△aprJ/ermE*p-tobO. c Growth state of Tb-△aprJ and Tb-△aprJ/ermE*p-tobO in MS solid medium. d Transcriptional analysis of the tobO in Tb-△aprJ, Tb-△aprJ/ermEp*-tobO, and Tb-△aprJ/kasOp*-tobO at 24 h, 72 h and 120 h in fermentation. e Transcriptional analysis of the tobB, tobE, tobM1, tobS1, tobT, and tobZ in Tb-△aprJ and Tb-△aprJ/ermEp*-tobO at 72 h in fermentation. Mean values of 3 replicates are shown, with the standards indicated by error bars

Fig. 9

Carbamoyltobramycin production measured by HPLC analysis. Mean values of 3 replicates are shown with the standard deviation as the error bars