Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Gao, Hong; Smith, Margaret C. M.

doi:10.1099/acmi.0.000291

Cited by 3 publications

(2 citation statements)

References 25 publications

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Section: Discussionmentioning

confidence: 99%

“…Recently, several groups have developed different ways of using multiple LSIs for DNA manipulation in vivo, [49] for example, using TG1, SV1, and ϕBT1 integrases to mediate multiplex conjugation and integration of plasmids from E. coli into Streptomyces. Fayed et al [50] used multiple integrases for in vivo assembly of the polyketide synthase genes to synthesize erythromycin in a streptomyces strain.…”

Section: Orthogonal Lsis Deliver High Fidelity Sira In a One-pot Reac...mentioning

confidence: 99%

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High fidelity one‐pot DNA assembly using orthogonal serine integrases

Abioye

Lawson-Williams

Lecanda

et al. 2022

Biotechnology Journal

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Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. Methods and Major Results:In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. Conclusions and Implications:We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.

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Section: Discussionmentioning

confidence: 99%

Section: Orthogonal Lsis Deliver High Fidelity Sira In a One-pot Reac...mentioning

confidence: 99%

High fidelity one‐pot DNA assembly using orthogonal serine integrases

Abioye

Lawson-Williams

Lecanda

et al. 2022

Biotechnology Journal

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Glucose-1-phosphate thymidylyltransferase promotes the production of 3-O-α-mycarosylerythronolide B in Streptomyces coelicolor

Gao,

Langer,

Larson

et al. 2024

Journal of Applied Microbiology

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Aims The main objective of this study was to produce erythronolide B (EB) and 3-O-α-mycarosylerythronolide B (MEB) in Streptomyces coelicolor and enhance the MEB production by expressing the glucose-1-phosphate thymidylyltransferase (RfbA). Methods and results We expressed eryF and eryB genes (eryBII, eryBIII, eryBIV, eryBV, eryBVI and eryBVII) to produce EB and MEB. The expression was confirmed by quantitative real-time PCR. Furthermore, the MEB's production was improved by more than 100-fold by expressing an enzyme, RfbA, which is absent from the erythromycin gene cluster, to promote the biosynthesis of TDP-L-mycarose. We discuss the feasibility of alternative Streptomyces species for erythromycin production based on the presence or absence of RfbA. Conclusions The RbfA enzyme from Saccharopolyspora erythraea was expressed in S. coelicolor M1152 along with the MEB biosynthesis pathway, resulting in a large increase in MEB production (>100-fold).

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Improvement of the conjugation transfer of N. gerenzanensis based on the synergistic effect of quorum sensing and antibiotic interference

shi,

Cheng,

Wang

et al. 2023

AMB Expr

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Nonomuraea gerenzanensis (N. gerenzanensis) is known for its ability to biosynthesize A40926, the precursor of the glycopeptide antibiotic (GPA) Dalbavancin. However, challenges and uncertainties related to the genetic manipulation of the rare actinomycetes remain. In order to improve the conjugation transfer of N. gerenzanensis, the crucial factors affecting conjugal transfer were evaluated, including agar medium, mycelial state, donor-recipient ratio, magnesium ion concentration, and antibiotic coverage time firstly. Additionally, γ-butyrolactone (GBL) for quorum sensing (QS) and antibiotics targeting bacterial walls were applied to evaluate their effects on conjugation transfer. As a result, the optimal conditions of 5%TSB of liquid medium, 24 h of the period time, V0.1 of agar medium, 30 mM of magnesium ion, the ratio 10:1 of donor-to-recipient, and 27 h of the overlaying time of antibiotic were determined. Furthermore, the results showed that autoinducer GBL and GPA teicoplanin had a synergetic effect on the conjugation transfer of N. gerenzanensis at a working concentration of 60 µM and 0.5 µg mL−1, respectively. The highest conjugation efficiency could reach about 1.3 depending on the optimal process conditions and the interference of QS and antibiotics.

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Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Cited by 3 publications

References 25 publications

High fidelity one‐pot DNA assembly using orthogonal serine integrases

High fidelity one‐pot DNA assembly using orthogonal serine integrases

Glucose-1-phosphate thymidylyltransferase promotes the production of 3-O-α-mycarosylerythronolide B in Streptomyces coelicolor

Improvement of the conjugation transfer of N. gerenzanensis based on the synergistic effect of quorum sensing and antibiotic interference

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