Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

Abstract

Background: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential.

Results: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed.

Conclusions: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Keywords: Antibiotic marker-free; Heterologous protein expression; Separate-component-stabilization system; Streptomyces; Toxin-antitoxin.

Graphical abstract

Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Fig. 1

Diagram of the separate component-stabilization system in Streptomyces. 1. Integration of the toxin (yoeBsl) gene into the chromosome of the S. lividans ∆TA (pGM160-YefMsl^ts) strain [22] with plasmid pTES-Tox. 2. Transformation with pALCre^ts to generate S. lividans ∆TA-pTES-Tox (pGM160-YefMsl^ts, pALCre^ts). 3. Elimination of apramycin resistance gene by induction of Cre recombinase. 4. Transformation with the expression plasmid (pNRoxAnti-Prot) and removal of the temperature-sensitive plasmids. 5. Transformation with pALDre^ts and elimination of the neomycin resistance gene from the expression plasmid (pNRoxAnti-Prot) by Dre recombinase induction. 6. Removal of the temperature-sensitive plasmid pALDre^ts and generation of the final host strain [S. lividans ∆TA -Tox (pRoxAnti-Prot)]. 7. Protein production

Fig. 2

Elimination of the apramycin resistance gene in the host strain genome. a Diagram of the integrative pTES-Tox plasmid. b Diagram of the pTES-Tox plasmid backbone deletion by the action of Cre recombinase (modified from [33]). c PCR amplification of the toxin gene (Tox) with primers LS-008 and LS-009 (1) and the apramycin resistance gene (Apra) with primers LS-113 and LS-114 (2) from S. lividans genomic DNA before, S. lividans ∆TA-pTES-Tox (−), and after, S. lividans ∆TA-Tox (+), Cre activity

Fig. 3

Amylase and Xylanase production by the different strains of S. lividans. a, c Diagram of the expression plasmids pNRoxAnti-Amy and pNRoxAnti-Xyl. b, d Amylase (b) and xylanase (d) production by S. lividans wt, S. lividans ΔTA and S. lividans ΔTA-Tox transformed with pNRoxAnti-Amy (b) and pNRoxAnti-Xyl (d) after 6 days of culture in YES medium supplemented with 3% xylose. 10 µL of the supernatant was loaded into each track

Fig. 4

Elimination of Neomycin gene in the expression plasmid. a Diagram of the deletion of neomycin resistance gene from the expression plasmid by Dre recombinase. b, c PCR and restriction analysis of pNRoxAnti-Amy and pRoxAnti-Amy (b) or pNRoxAnti-Xyl and pRoxAnti-Xyl (c). 1. Neo: Primers LS124 and LS125. 2. Amy: Primers MRG11 and MRG12. 3. Xyl: Primers LS116 and LS117. The arrow shows the neomycin restriction band. d, e Amylase and xylanase production by S. lividans ΔTA-Tox transformed with pNRoxAnti-Amy and pRoxAnti-Amy (d) or pNRoxAnti-Xyl and pRoxAnti-Xyl (e) after 6 days of culture in YES medium supplemented with 3% xylose. 10 µL of the supernatant was loaded into each track

Fig. 5

Enzyme production over the time of culture. a, c Amylase and xylanase production by S. lividans ΔTA-Tox transformed with pRoxAnti-Amy (a) and pRoxAnti-Xyl (c) after 2, 4, 6 and 8 days of culture in YES medium supplemented with 3% xylose. 10 µL of the supernatant was loaded into each track. b, d Amylase (b) and xylanase (d) activity of the supernatants. The histogram bars are the means of three experiments

Fig. 6

Enzyme production after strains storage and after serial subcultures. a and c Amylase and xylanase production by S. lividans ΔTA-Tox transformed with pRoxAnti-Amy (a) and pRoxAnti-Xyl (c) after mycelia storage (M) and after sporulation (S). 10 µL of the supernatant collected after 6 days was loaded into each track. b and d Percentages of amylase (b) and xylanase (d) activity of the supernatants compared with the original culture (1). e and g Amylase and xylanase production by S. lividans ΔTA-Tox transformed with pRoxAnti-Amy (e) and pRoxAnti-Xyl (g) after three 100-fold serial dilutions every 2 days (P1, P2, and P3) in fresh YES medium supplemented with 3% xylose. 10 µL of supernatants collected after 6 days was loaded into each track. f and h percentage of amylase (f) and xylanase (h) activity of the supernatants. The histogram bars are the mean of three experiments