基于pyrF作为反选标记构建适用于龟裂链霉菌的基因敲除系统

Development of a pyrF -based counterselectable system for targeted gene deletion in Streptomyces rimosus 基于 pyrF 作为反选标记构建适用于龟裂链霉菌的基因敲除系统 1 equal first-author 2 equal 1 2 1 1 1 1 2 2 2 2 2 3 3 2 2 2 corresp Zilong LI, lizl@im.ac.cn Zilong LI, lizl@im.ac.cn lizl@im.ac.cn https://orcid.org/0000-0002-4568-6902 1 corresp corresp Zilong LI, lizl@im.ac.cn Zilong LI, lizl@im.ac.cn Shouliang YIN, yinsl@ncst.edu.cn Shouliang YIN, yinsl@ncst.edu.cn yinsl@ncst.edu.cn https://orcid.org/0000-0001-5087-3289 School of Life Sciences, North China University of Science and Technology, Tangshan063210, China School of Life Sciences, North China University of Science and Technology, Tangshan063210, China State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing100101, China State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing100101, China Shengxue Dacheng Pharmaceutical Co., Ltd., Shijiazhuang051430, China Shengxue Dacheng Pharmaceutical Co., Ltd., Shijiazhuang051430, China Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (Δ pyrF ) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant Δ pyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF- based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species. 目的 2 以 pyrF 作为反选标记基因构建基因敲除系统，快速、高效敲除龟裂链霉菌的靶基因。 创新点 2 在链霉菌种属内首次采用 pyrF 作为反选标记基因，以5-氟乳清酸作为筛选因子，实现靶基因的敲除。与传统的敲除方法相比，该敲除系统获得同源双交换突变株的时间大大缩短。 方法 2 基于 pyrF 反选标记的基因敲除系统包括一个尿嘧啶营养缺陷型宿主（Δ pyrF ）和一个携带互补基因 pyrF 的非自主复制型的敲除载体（pKC1132-pyrF）。第一步，将携带有互补基因 pyrF 的敲除质粒导入到尿嘧啶营养缺陷型宿主（Δ pyrF ）中，只有发生单交换重组的突变株，才能在缺少尿嘧啶的基本培养基中生长；第二步，将发生单交换的突变株接种到含有5-氟乳清酸的基本培养基中，只有发生第二次同源重组，且被敲除的靶基因、 pyrF 基因及质粒骨架发生重组丢失的突变株才能在含有5-氟乳清酸的培养基中生长。经过两步筛选，快速、高效实现靶基因的敲除。 结论 2 本文通过生物信息学比对、基因敲除和回补实验验证 pyrF 基因为乳清酸核苷-5’-磷酸脱羧酶的编码基因。利用 pyrF 基因作为反选标记，以5-氟乳清酸作为筛选因子，快速、高效的实现了龟裂霉菌的靶基因 otcR 的敲除。本文构建的基因敲除系统为链霉菌的分子遗传操作提供了一种新的遗传工具。 Counterselectable system pyrF 5-Fluoroorotic acid (5-FOA) Gene deletion Streptomyces rimosus 反选敲除系统 pyrF 5-氟乳清酸 基因敲除 龟裂链霉菌 1 Introduction 1 Streptomyces is a genus of more than 900 species of filamentous actinobacteria, most of which can produce many kinds of antibiotics and other natural bioactive products ( Barka et al., 2016 Barka et al., 2016 ; Wang et al., 2020 Wang et al., 2020 ; Xia et al., 2020 Xia et al., 2020 ). However, the increasing occurrence of multidrug resistant bacteria is creating an urgent need to increase the yield of approved natural drugs and to discover more novel classes of antibiotics with remarkable antimicrobial effects. Modern genome sequencing, alignments, and annotations of Streptomyces species have revealed that most natural products encoded in their genomes remain undiscovered, because of numerous cryptic secondary metabolite gene clusters encoding products that are either silent or not identified as natural products during growth in the laboratory ( Niu et al., 2016 Niu et al., 2016 ; Li et al., 2019 Li et al., 2019 ). Novel and efficient gene editing tools for the genetic manipulation of Streptomyces genes are highly desired to exploit these treasure troves ( Zeng et al., 2015 Zeng et al., 2015 ). The pyrimidine biosynthesis pathway is highly conserved from bacteria to humans and provides an interesting target for molecular genetic manipulation due to the selection role of the bactericidal pyrimidine analog 5-fluoroorotic acid (5-FOA) ( Yano et al., 2005 Yano et al., 2005 ; Capone et al., 2007 Capone et al., 2007 ). Orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10, encoded by pyrE ) catalyzes the conversion of orotic acid to the normal product orotidine-5'-monophosphate (OMP), but 5-FOA used as substrate is converted to form 5‐fluoroorotidine-5'-monophosphate (5-FOMP) ( Fig. 1 Fig. 1 ). 5-FOMP is converted into the product 5-fluorouridine-5'-monophosphate (5-FUMP) instead of uridine-5'‐monophosphate (UMP) by the catalytic action of orotidine-5'-monophosphate decarboxylase (OMPDCase, EC 4.1.1.23, encoded by pyrF ), which catalyzes a key step in de novo pyrimidine biosynthesis ( Brockman et al., 1960 Brockman et al., 1960 ; West, 2012 West, 2012 ). Although 5-FOA is not directly responsible for toxicity, the accumulation of cellular components containing 5-FUMP often leads to cell death due to their toxicity ( O'Donovan and Neuhard, 1970 O'Donovan and Neuhard, 1970 ; Boeke et al., 1984 Boeke et al., 1984 ; Kurniyati and Li, 2016 Kurniyati and Li, 2016 ). Thus, mutants with deletion of the pyrF gene are uracil auxotrophs but resistant to 5-FOA, creating an opportunity whereby the ectopic expression of pyrF with a non-replicative plasmid can be selected or counterselected ( Boeke et al., 1984 Boeke et al., 1984 ; D'Enfert, 1996 D'Enfert, 1996 ). The pyrF -based gene disturbance system has been successfully adapted in various organisms, such as yeasts ( Staab and Sundstrom, 2003 Staab and Sundstrom, 2003 ), fungi ( Fujii et al., 2012 Fujii et al., 2012 ), archaea ( Liu et al., 2011 Liu et al., 2011 ), and other bacterial species. However, to the best of our knowledge, no study has reported the application of pyrF -based counterselectable gene knockout systems to Streptomyces species. The aim of this study was to explore the potential for using pyrF as a counterselectable marker to establish an efficient screening system in Streptomyces species. The genetic manipulation of Streptomyces strains is considerably more difficult than that of other traditional model microorganisms, such as Escherichia coli and Saccharomyces cerevisiae , because few gene knock-in and knock-out methods are available ( Zhao et al., 2018 Zhao et al., 2018 ). Few genetic manipulation tools work well in industrial Streptomyces strains ( Liu et al., 2018 Liu et al., 2018 ). Industrial strains that have undergone multiple rounds of exposure to ultraviolet (UV) radiation or chemical mutagens (e.g. nitrosoguanidine) are often resistant to several antibiotics; hence, no suitable antibiotic-selection marker is available for genetic manipulation. Streptomyces rimosus is characterized as the best industrial streptomycete producer of oxytetracycline (OTC) and other tetracycline antibiotics, with the highest OTC production yield of 35 g/L in large-scale fermentations. However, genetic modification of S. rimosus is considerably more difficult than that of other species due to its intrinsic high resistance to many commonly used antibiotics ( Song et al., 2019 Song et al., 2019 ). The minimal inhibitory concentrations in S. rimosus M4018 are 1000 µg/mL for spectinomycin, 400 µg/mL for kanamycin, and 500 µg/mL for apramycin ( Yu et al., 2012 Yu et al., 2012 ; Yin et al., 2016 Yin et al., 2016 , 2017 2017 ; Wang et al., 2019 Wang et al., 2019 ). Therefore, most gene disruption tools are inefficient due to the lack of suitable selectable markers and a low homologous recombination (HR) frequency in industrial microbiology. Conventional gene editing in Streptomyces is based mainly on HR using a double-crossover integration, site-specific recombination system, or I- Sce I endonuclease system with an antibiotic selection marker. Usually, hundreds of clones must be screened, requiring a multistep process or leaving a recombinase target site in the chromosome to select the desired mutants, resulting in a time-consuming or arduous process ( Zeng et al., 2015 Zeng et al., 2015 ; Niu et al., 2016 Niu et al., 2016 ; Kong et al., 2019 Kong et al., 2019 ). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is a powerful gene-editing technology with high efficacy for site-specific gene targeting. The application of the CRISPR/Cas9 system to genome editing in Streptomyces is highly developed. Diverse approaches are widely used to edit or refactor biosynthetic gene clusters for natural drug discovery and characterization ( Jia et al., 2017 Jia et al., 2017 ; Tao et al., 2018 Tao et al., 2018 ). However, potential off-target effects have raised major concerns regarding their application ( Zhang et al., 2015 Zhang et al., 2015 ). In this study, we developed a pyrF -based gene disruption screening system in S. rimosus M4018 and demonstrated its efficient application to targeted gene deletion. This new system should facilitate genetic manipulation of other Streptomyces species. 2 Materials and methods 1 2.1 Strains and culture conditions 2 The strains and plasmids used in this study are listed in Table 1 Table 1 . E. coli strain DH5α (Invitrogen, Waltham, Massachusetts, USA) was used for plasmid DNA manipulation; E. coli ET12567/pUZ8002 was used as a host for transferring DNA from E. coli to Streptomyce s by conjugation ( Kieser et al., 2000 Kieser et al., 2000 ). E. coli strains were grown in Luria-Bertani (LB) medium supplemented with chloramphenicol (25 μg/mL), kanamycin (25 μg/mL), or apramycin (50 μg/mL) when necessary. The S. rimosus transformants were screened and selected on minimal medium (MM) plates containing nalidixic acid (25 μg/mL) and apramycin (500 μg/mL) when necessary. Spores of S. rimosus were obtained on mannitol soya flour (MS) medium ( Kieser et al., 2000 Kieser et al., 2000 ) after incubation at 30 ℃ for 5 d. 5-FOA and uracil were purchased from the Sigma Chemical Company (St. Louis, USA) and solubilized in dimethyl sulfoxide (DMSO) before being added to the growth medium. DNA restriction endonucleases and DNA polymerases (Phusion, Q5, and Taq) were purchased from New England Biolabs, Inc. (NEB, Ipswich, Massachusetts, USA). The other chemicals and reagents, unless noted, were obtained from local commercial sources. Name Description Source Streptomyces   S. rimosus M4018 Used for commercial production of oxytetracycline (OTC) Tang et al., 2011 Tang et al., 2011  Δ pyrF pyrF gene disruption mutant, derived from M4018 This study  Δ pyrF::pyrF pyrF complemented strain, derived from Δ pyrF This study  Δ pyrF Δ otcR otcR gene disruption mutant, derived from Δ pyrF This study  Δ pyrF Δ otcR::otcR otcR complemented strain, derived from Δ pyrF Δ otcR This study Escherichia coli  DH5α General cloning host for plasmid manipulation Invitrogen  ET12567 (pUZ8002) Donor strain for conjugation between E. coli and Streptomyces Kieser et al., 2000 Kieser et al., 2000 Plasmids  pKC1132 Conjugative vector, Am R ( aac(3)IV ), lacZ , ori pUC18 , oriT Yin et al., 2015 Yin et al., 2015  pKC1132-pyrFDM For deletion of the pyrF gene, containing upstream and downstream homologous arms of pyrF This study  pSET152 E. coli - Streptomyces shuttle vector, Am R ( aac(3)IV ), lacZ , ori pUC18 , attp ( φC31 ), oriT , and int Bierman et al., 1992 Bierman et al., 1992  pSET152-pyrF Conjugative, integrative vector derived from pSET152, containing the pyrF gene driven by the promoter of SF14 This study  pKC1132-pyrF Conjugative, derived from pKC1132, containing the pyrF gene driven by the promoter of SF14 This study  pOtcRDM Conjugative vector, containing upstream and downstream homologous arms of otcR Yin et al., 2015 Yin et al., 2015  pKC1132-otcRDM1 Conjugative, derived from pKC1132-SFpyrF, containing upstream and downstream homologous arms of otcR This study  pSF14-otcR Derived from pGusT-SF14, SF14-driven otcR Yin et al., 2015 Yin et al., 2015 2.2 Construction of plasmids 2 All the primers used in this work are listed in Table 2 Table 2 . The introduced overlapping sequences used for Gibson assembly are shown in bold. Name Sequence (5' to 3') Use pyrF-UP-F CGACGTTGTAAAACGACGGCCAGTGCCAAGCTT CGTCCTACGACGAGGAGAGCG 4 Construction of pKC1132-pyrFDM pyrF-UP-R TCGTCGGCACATCGGACTGCAG GAAGGCGGACTGCGGCTTGAG pyrF-DN-F AAGCCGCAGTCCGCCTTCCTGCAG TCCGATGTGCCGACGAGGTGC pyrF-DN-R TTACGAATTCGATATCGCGCGCGGCCGCGGATC CCCGATCATGAGGTAGGAGG Test1-F GAGCACAAGAAGGTGACCGC 2 Identification of the recombinant Δ pyrF Test1-R TGACGTCATGAAGGGAGGCG ExTest1-F TGGCTGATCATTGGCGGAAG 2 Identification of the recombinant Δ pyrF Extest1-R TCGTCGACCAGCTCTTCCTG otcR-UPDN-F GCCGCCTCATCAAGGTCAAGGC TCTAGAGTGGCCATTGCGGACTTCTACC 2 Construction of pKC1132-otcRDM1 otcR-UPDN-R GCCGTACCTCTACGACTACTTGAATTC GTAATCATGTCATAGCTGTTTCC Test2-F CAGAGGGACATATTGCCGCC 2 Identification of the recombinant Δ pyrF Δ otcR Test2-R TCATGACCTGGTTCCTGCGC SFpyrF1-F CACAGAACCACTCCACAGGAGGACCCATATG ACGGCCCCGACCCCCTTCG 4 Construction of pKC1132-pyrF SFpyrF1-R CGGCCCTGGTTCCTTCTTGTTGTCTAGA CTAAAGTCCCCGTCGTTCGACCGC SFpyrF2-F GTTGTAAAACGACGGCCAGTGCCAAGCTT CTAAAGTCCCCGTCGTTCGAC SFpyrF2-R GATATCGCGCGCGGCCGCGGATCCTCTAGA GCCTTGACCTTGATGAGGCG 2.2.1　Construction of the vector for deletion of pyrF 3 To construct the pyrF deletion vector plasmid pKC1132-pyrFDM, the upstream homologous arm fragment (3080 bp) of pyrF was amplified with primers pyrF-UP-F/pyrF-UP-R, and the downstream homologous arm fragment (3237 bp) was amplified with primers pyrF-DN-F/pyrF-DN-R from the genomic DNA template of M4018. These two fragments were joined with pKC1132 (digested with Bam HI/ Hin dIII) using the recombination method described by Gibson et al. (2009) Gibson et al . (2009) . To construct the complementation plasmid pSET152-pyrF, the pyrF fragment was amplified with primers SFpyrF1-F/SFpyrF1-R from the template of genomic DNA, and inserted into the Nde I/ Xba I sites of pSF14-otcR following the Gibson assembly method. 2.2.2　Construction of the non-replicative and pyrF -complementary plasmid for deletion of otcR 3 A 1029-bp fragment containing both the SF14 promoter and pyrF was amplified from the template pSET152-pyrF with primers SFpyrF2-F/SFpyrF2-R. Subsequently, this fragment was inserted into the Hin dIII/ Xba I sites of pKC1132 to generate plasmid pKC1132-pyrF. To construct the otcR disruption plasmid pKC1132-otcRDM1, the upstream and downstream fragments were amplified from pOtcRDM using primers otcR-UPDN-F/otcR-UPDN-R, and then inserted into the Eco RI/ Xba I sites of pKC1132-pyrF. 2.3 Deletions of pyrF and otcR 2 The plasmid pKC1132-pyrFDM was introduced into the strain M4018 to obtain apramycin-resistant transconjugants that had integrated pKC1132-pyrFDM into the chromosome as a result of one crossover. Then, the transconjugants were subcultured in MM medium with 100 μmol/L 5-FOA and 200 μmol/L uracil, and grown for 4 d at 30 ℃ to select against the pyrF wild-type (WT) allele. The pyrF gene was eliminated along with the non-replicative plasmid skeleton from the chromosome as a result of a second crossover. The extracted DNA samples were then subjected to polymerase chain reaction (PCR) amplification. The correct isolation of Δ pyrF was confirmed using internal primers Test1-F/Test1-R (found on both the plasmid and the genome). The PCR program included: initial denaturation (5 min, 95 ℃) followed by 30 cycles of denaturation (30 s, 95 ℃), annealing (30 s, 56 ℃), elongation (1.5 min, 68 ℃), and then final extension (10 min, 68 ℃). The disruption of pyrF was also confirmed by external primers ExTest1-F/ExTest1-R (found only in the genome). The PCR elongations were performed at 68 ℃ using the extension time of 7.5 min (1 min per 1000 bp). Deletion of the otcR gene was performed using the pyrF -based gene knockout systems. Briefly, the Δ pyrF strain conjugated with pKC1132-otcRDM1 ( Table 1 Table 1 ) was grown in uracil-free MM medium for about 3 to 4 d to select for uracil prototrophy. Then, to screen the transconjugants of Δ pyrF Δ otcR mutants, the above uracil auxotrophic strains were collected and cultured using solid MM medium supplemented with 100 µmol/L 5-FOA and 200 μmol/L uracil. All the 5-FOA resistant colonies were screened by PCR using the primers Test2-F/Test2-R ( Table 2 Table 2 ). PCR extensions were normally performed at 68 ℃ using the extension time of 1.5 min. 2.4 Bioinformatic analysis 2 The selected putative OMPDCase sequences from Streptomyces were aligned by Clustal Omega (Conway Institute UCD Dublin, Dublin, Ireland) ( Sievers et al., 2011 Sievers et al., 2011 ). The sequence similarities and secondary structure information from aligned sequences were rendered and displayed with the server ESPript 3.0 ( https://espript.ibcp.fr/ESPript/ESPript/index.php https://espript.ibcp.fr/ESPript/ESPript/index.php ) ( Robert and Gouet, 2014 Robert and Gouet, 2014 ). 2.5 Determination of 5-FOA susceptibility 2 All the strains were incubated on MS medium plates at 30 ℃ for 5 d, and then the spores were collected and diluted with sterilized water to a final concentration from 1×10 6 to 1×10 3 mL -1 using plate colony-counting methods. Afterwards, 5 μL of the spore suspension was spread on MM culture plates supplemented with increasing amounts of 5-FOA and uracil (0 and 0, 0 and 250, 250 and 250, 500 and 250 μmol/L, respectively). The growth and morphology of the colonies incubated on the plates were used to evaluate the tolerance of the strains to 5-FOA. 2.6 Fermentation and detection of OTC production 2 The spore suspensions (the total number of spores is 1.0×10 8 ) of S. rimosus were inoculated into 40 mL of R5 medium with 1 mmol/L uracil on a rotary shaker (250 r/min) for 24 h at 30 ℃, and then 2 mL of the seed cultures were transferred into 40 mL fresh R5 medium and incubated at 30 ℃ for 4 d. The OTC production was quantified by high-performance liquid chromatography (HPLC), as previously described ( Yin et al., 2015 Yin et al., 2015 ). First, the fermentation samples were adjusted to pH 1.5-2.0 with 9 mol/L HCl, and 1-mL samples were centrifuged at 12 000 r/min for 10 min. Then, the supernatants were subjected to HPLC analysis on a Shimadzu Prominence HPLC system (Kyoto, Japan) with a dual λ UV detector and YMC polymer C18 column (4.6 mm×250 mm; Kyoto, Japan). Separation was performed with the following conditions: 60% H 2 O, 10% methanol, 20% acetonitrile, and 10% phosphoric acid (2 mmol/L) at a constant flow rate of 1 mL/min. The OTC peak areas detected at 350 nm were used to calculate the concentration of OTC. 3 Results 1 3.1 Sensitivity of Streptomyces strains to 5-FOA 2 Gene knockout systems based on pyrF as the selection marker have been successfully applied to Treponema denticola ( Kurniyati and Li, 2016 Kurniyati and Li, 2016 ), Shewanella livingstonensis ( Ito et al., 2016 Ito et al., 2016 ), Caldicellulosiruptor hydrothermalis ( Groom et al., 2014 Groom et al., 2014 ), Haloarcula hispanica and Haloferax mediterranei ( Liu et al., 2011 Liu et al., 2011 ), Candida albicans ( Staab and Sundstrom, 2003 Staab and Sundstrom, 2003 ), and some other bacterial species. However, no study has reported the application of this system to Streptomyces . To develop a pyrF -based selection and counter-selection system, the WT strain must be sensitive to 5-FOA, and the strain with deletion of pyrF must confer resistance to 5-FOA. Thus, as an initial step, we tested the sensitivities of Streptomyces coelicolor M145, Streptomyces venezuelae ISP 5230, S. rimosus M4018, and Streptomyces aureofaciens ATCC 10762 to 5-FOA. The cells grew well in MM medium without supplementation of 5-FOA ( Fig. 2 Fig. 2 ). However, 5-FOA at a concentration of 5 μmol/L inhibited the growth of S. coelicolor M145 and S. venezuelae ISP 5230, and the growth of all Streptomyces strains was completely inhibited when the concentration reached 50 μmol/L. These results indicated that Streptomyces strains were extremely susceptible to 5-FOA, and therefore highly likely to be good candidates for the development of an efficient gene disruption system using 5-FOA as the screening factor. 3.2 Identification of OMPDCase and creation of S. rimosus Δ pyrF host 2 The sensitivity to 5-FOA indicates the presence of genes related to the pyrimidine synthesis pathway in Streptomyces . The OMPDCase ortholog ( pyrF ) was annotated on the genome of S. rimosus provided by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov ). All the information corresponding to the protein, source, and database accession number was given by a search with the basic local alignment search tool (BLAST). The deduced PyrF polypeptide (GenBank No. ELQ81494.1) from S. rimosus exhibits high similarity with those of a wide range of Streptomyces species, such as S. avermitilis (GenBank No. KUN53591.1), S. coelicolo r (GenBank No. TYP05411.1), S. venezuelae (GenBank No. WP_150188059.1), and S. aureofaciens (GenBank No. WP_030290267.1) ( Fig. 3 Fig. 3 and Table S1) ( Chenna et al., 2003 Chenna et al., 2003 ; Robert and Gouet, 2014 Robert and Gouet, 2014 ). The crystal structure of putative OMPDCase from S. avermitilis (protein data bank (PDB) accession number: 3V75) comprises 12 α‐helices (48.21% of amino acid, 135 residues) and 8 β‐strands (17.86% of amino acid, 50 residues). Interestingly, the putative OMPDCase of Streptomyces exhibits low similarity with the PyrF (GenBank No. ACT29365.1) of E . coli , which contains a 265-amino acid polypeptide ( Table 3 Table 3 ). Multiple sequence alignment through Clustal Omega revealed that the OMPDCase amino acid sequence of E. coli exhibits about 20% sequence pair identity and less than 30% similarity with that of Streptomyces species ( Sievers et al., 2011 Sievers et al., 2011 ). Source Amino acid residue GenBank accession No. Similarity (%) Identity (%) Escherichia coli 265 ACT29365.1 Streptomyces avermitilis 280 KUN53591.1 29.8 20.8 Streptomyces coelicolor 281 TYP05411.1 28.7 19.9 Streptomyces venezuelae 278 WP_150188059.1 26.5 19.5 Streptomyces rimosus 286 ELQ81494.1 31.0 19.3 Streptomyces aureofaciens 277 WP_030290267.1 23.0 16.7 Alignment analysis of the whole genome sequence revealed that only one copy of the putative OMPDCase gene pyrF exists on the genome of S. coelicolor A3, S. venezuelae ISP 5230, S. rimosus M4018, and S. aureofaciens ATCC 10762. In the genome of S. rimosus M4018, pyrF is encoded in an operon between a conserved hypothetical protein and a predicted dihydroorotate dehydrogenase. Fig. 4 Fig. 4 a shows a schematic of the strategy used to delete the pyrF gene in S. rimosus . The pKC1132-pyrFDM vector was constructed ( Fig. 4 Fig. 4 a) and conjugated into the WT strain S. rimosus M4018 as described in Section 2.2.1. The suicide plasmid, pKC1132-pyrFDM, which contained the apramycin resistance genes and the pyrF flanking sequences, was introduced into the WT strain S. rimosus M4018. Plasmids harboring recombinants of a single-crossover were selected on apramycin plates. Through plating the apramycin-resistant transconjugants on MM medium containing 100 μmol/L 5-FOA and 200 μmol/L uracil, the pyrF gene was eliminated along with the non-replicative plasmid skeleton from the genome of M4018 by the second HR. The Δ pyrF strain was generated and selected by 5-FOA ( Fig. 4 Fig. 4 a), because PyrF is responsible for uracil biosynthesis and its deletion confers resistance to 5-FOA ( Fig. 4 Fig. 4 b). Among the 50 colonies resistant to 5-FOA, 49 strains were found to be uracil auxotrophs. The correct construction of the Δ pyrF mutant was confirmed by PCR using primers Test1-F/Test1-R, where the 600-bp difference between these DNA fragments of the WT strain and the Δ pyrF strain was consistent with the loss of pyrF (Figs. 4a 4a and 4c 4c ). The disruption of pyrF was also confirmed by external primers ExTest1-F/ExTest1-R (found only in the genome). The length of predicted product detected by PCR was 6640 bp (Δ pyrF ) instead of 7140 bp (M4018), indicating that the pyrF gene had been deleted from the chromosome (Fig. S1). 3.3 Characterization and complementation of the S. rimosus Δ pyrF strain 2 The WT, Δ pyrF , and pyrF -complementing (Δ pyrF::pyrF ) strains were tested on solid uracil-free defined medium with or without supplementation of 5-FOA and uracil, to evaluate the characteristics of 5-FOA sensitivity/resistance and uracil auxotrophy/prototrophy. The Δ pyrF strain could not grow well in synthetic MM medium without uracil because of the incomplete pyrimidine synthesis pathway, whereas the growth of parental and pyrF -complementing strains was uninhibited ( Fig. 5 Fig. 5 ). The Δ pyrF strain proliferated when supplemented with 250 µmol/L uracil, and the morphology and sporulation of the clones were similar to those of the WT strain. Thus, deletion of the pyrF gene did not hamper growth when enough uracil was provided ( Fig. 5 Fig. 5 ). The pSET152-pyrF plasmid was transformed and integrated into the pyrF mutant at the φ C31-mediated site-specific integration locus to reintroduce the pyrF gene into the genome of the Δ pyrF mutant. The pyrimidine biosynthetic pathway in the mutant Δ pyrF was complemented and restored as in the parental strain by constitutive expression of the OMPDCase gene. When the concentration of 5-FOA was increased, the pyrF -complementing strain and parental strain became sensitive to 5-FOA, and their growth was completely suppressed, whereas the mutants (Δ pyrF ) lacking pyrF could still grow well in the presence of 5-FOA, indicating that deletion of pyrF conferred 5-FOA resistance in the mutants. All these results confirmed that the OMPDCase gene pyrF could be used as a counterselection marker in the Δ pyrF host. The catalytic activity of PyrF is required for 5-UMP toxicity, so the lack of toxicity under conditions of excess uracil likely reflects the activity of the salvage pathways of pyrimidine, possibly due to the downregulation of de novo pyrimidine synthesis when exogenous uracil is available ( Capone et al., 2007 Capone et al., 2007 ). 3.4 Constitution and principle of the pyrF -based screening system 2 A pyrF -based gene knockout system should contain a uracil auxotrophic host (Δ pyrF ) and a non-replicative plasmid vector carrying the complementary gene pyrF , which could act as a positive selection and counterselection marker for generation of knockout mutants ( Tripathi et al., 2010 Tripathi et al., 2010 ). This non-replicative plasmid vector pKC1132-pyrF for HR was constructed as described in the Section 2.2.1. The vector included an apramycin resistance cassette ( aac(3)IV ), an OMPDCase gene pyrF from S. rimosus M4018 driven by the SF14 promoter, a segment of plasmid pKC1132 containing the origin of replication ( ori -colE1) from E. coli , an origin of plasmid DNA transfer element ( oriT ) for RK2-mediated mobilization, and multiple cloning sites for facilitating cloning of the desired gene ( Fig. 6 Fig. 6 a). First, a single-crossover transformant was constructed by reintroducing pyrF along with pKC1132-pyrF derivatives into the chromosome of the S. rimosus Δ pyrF host at the specific locus of the chosen target gene. The transformant can be obtained by plating spores on MM medium without uracil, because the obtained single-crossover mutation restores the uracil synthesis capability and susceptibility to 5-FOA. Next, given that only the targeted gene knockout mutants lacking PyrF activity can grow well in the medium containing 5-FOA, a clean marker-free mutant, from which the pyrF gene, antibiotic resistance marker, and the targeted gene have been excised through allelic exchange recombination, must be created to screen for 5-FOA-resistant colonies by using culture medium supplemented with 5-FOA. 3.5 Targeted gene deletion in S. rimosus with pyrF -based gene knockout systems 2 To demonstrate the feasibility of using the non-replicative plasmid pKC1132-pyrF in gene deletion in S. rimosus Δ pyrF , we used it to delete the gene otcR , identified in our previous research as a coding gene of a cluster-situated pathway specific activator for OTC biosynthesis ( Yin et al., 2015 Yin et al., 2015 ). The plasmid pKC1132-otcRDM1 ( Fig. 6 Fig. 6 b) containing two internal upstream and downstream fragments of otcR was introduced into S. rimosus Δ pyrF , and MM medium lacking uracil was used to select the Δ pyrF mutant clones restored to uracil prototrophy as a result of the transformation of plasmid pKC1132-otcRDM1 containing the pyrF gene by single crossover ( Fig. 7 Fig. 7 a). Counterselection for a pyrF excision with a double crossover, targeting gene otcR -knockout recombinants (Δ pyrF Δ otcR ) was performed by screening for 5-FOA-resistant colonies. More than 99% of the colonies screened by 5-FOA indicated the high accuracy of HR at the otcR locus. Strains in which otcR was knocked out were discernible by a PCR amplicon 862 bp shorter than that of the WT. The expected amplicon was 1462 bp for the S. rimosus Δ pyrF and 600 bp for the Δ pyrF Δ otcR strain ( Fig. 7 Fig. 7 b). OtcR as the activator of the OTC biosynthesis pathway is essential for OTC production in S. rimosus . OTC production was completely abolished in the fermentation broth of the Δ pyrF Δ otcR strain analyzed by HPLC. Meanwhile, complementation of the Δ pyrF Δ otcR mutant with a single copy of otcR (Δ pyrF Δ otcR::otcR ) restored the production of OTC to a level similar to that of the parental strain ( Fig. 7 Fig. 7 c). With the counterselection marker applied in this screening system, the time taken to obtain the double-crossover recombinants from single-crossover mutants was reduced from more than two weeks to about only one week. A series of concentration gradients were used to obtain the appropriate concentrations of 5-FOA and uracil for counterselection. We determined that concentrations of 5-FOA from 50 to 100 µmol/L were sufficient for inhibiting the growth of the WT strain and pyrF plasmid-curing mutants. When the concentration of uracil in the medium gradually increased from 100 and 200 µmol/L to 300 µmol/L, the desired otcR elimination strain grew from a "clear" to "not clear" background ( Figs. 4 Figs. 4 and S2). False positive clones ( pyrF -assisted plasmid was not lost) appeared and increased at high concentrations of uracil ( > 300 μmol/L) (Fig. S2). The surviving false positive clones could use the highly concentrated uracil via a salvage pathway and reduce the de novo synthesis pathway. The size of the predicted positive clones was large; however, the false-positive clones were very small, numerous ("not clear" background), and sensitive to 5-FOA after subculture. 4 Discussion 1 In this work, we developed a simple pyrF -based counterselectable system to allow efficient homologous gene disruption in S. rimosus . The de novo pyrimidine biosynthesis and salvage pathways are essential processes in organisms. If the required substrates in the surrounding environment are available and adequate, the salvage pathways are usually preferred over de novo synthesis. The pyrF gene encoding OMPDCase is involved in the de novo biosynthesis of pyrimidine-related metabolites, such as UMP, uridine-5'-diphosphate (UDP), and uridine-5'-triphosphate (UTP), and also can metabolize 5-FOA to toxic metabolites ( Suzuki et al., 2012 Suzuki et al., 2012 ). Deficiency in PyrF makes a mutant strain uracil auxotrophic and 5-FOA resistant. Thus, the Δ pyrF mutant was expected to allow the use of pyrF as a counterselection marker on a non-replicating or suicide plasmid with 5-FOA as the selection agent ( Capone et al., 2007 Capone et al., 2007 ). Most of the plasmids used for recombinant cloning technology in bacteria are derived by using antibiotic resistance genes as selection markers. However, given the sensitive issues of the appearance of antibiotic-resistant strains and legal requirements, these antibiotics must be avoided as much as possible in the industrial process. Thus, different antibiotic marker-free approaches, such as the complementation of auxotrophic strains, have been developed ( Vandermeulen et al., 2011 Vandermeulen et al., 2011 ; Sevillano et al., 2017 Sevillano et al., 2017 ). With the application of the pyrF -based counterselectable screening system, two steps are followed to obtain the desired mutant ( Fig. 7 Fig. 7 a). In the first step, a single recombination event is selected by uracil prototrophy. In the second step, a double recombination event is selected by resistance to 5-FOA ( Galvão and de Lorenzo, 2005 Galvão and de Lorenzo, 2005 ). With the assistance of this pyrF -screening marker, the non-replicative plasmid-integrated colonies can be rapidly selected by one-plate screening in a single step instead of the conventional multi-step continuous screening operation, and with a more certain outcome. This makes successive gene disruption highly accomplishable with improved stability and efficiency ( Tripathi et al., 2010 Tripathi et al., 2010 ). Another advantage of this screening system is that the genome is unmarked after pyrF selection and counterselection, so additional genetic manipulations with the same plasmid derivatives are possible and convenient. The regulation of pyrimidine de novo biosynthesis genes has been well studied in E. coli and Bacillus subtilis , and the attenuator PyrR can repress the expression of pyr genes at the transcription level ( Tanaka et al., 2015 Tanaka et al., 2015 ). To increase the sensitivity of 5-FOA, Suzuki et al. (2012) Suzuki et al. (2012) constructed the double mutant strain Δ pyrF Δ pyrR in Geobacillus kaustophilus , which is prototrophic for uracil and 5-FOA sensitive. This double-knockout strain (Δ pyrF Δ pyrR ) was constructed because the single-gene deletion mutant Δ pyrF is auxotrophic for uracil and resistant to 5-FOA. This complementary strain with pyrF is prototrophic for uracil, but still insensitive to 5-FOA in the presence of uracil ( Suzuki et al., 2012 Suzuki et al., 2012 ). In our study, to reduce the inhibitory effect of PyrR and improve the screening efficiency, we deliberately used the non-replicative vector containing pyrF driven by the constitutively expressed and stronger promoter SF14 instead of its native promoter ( Labes et al., 1997 Labes et al., 1997 ; Wang et al., 2013 Wang et al., 2013 ). In our study, the frequencies of 5-FOA-resistant clones and false positives were analyzed in S. rimosus using the proposed screening system for the deletion of otcR . The pyrF -based counterselection system was very effective and enabled us to obtain the desired mutant by analyzing only a small number of clones. The screening rate of positive clones was higher than that of the traditional method. The frequencies of false positives and revertants could be significantly reduced by adding appropriate proportions of uracil (200 μmol/L) and 5-FOA (100 µmol/L) to the MM medium. In summary, we successfully constructed a novel genetic manipulation system to perform targeted gene manipulation in S. rimosus . When counterselection was applied in this pyrF -based system, double-crossover recombinants were obtained from single-crossover recombinants much more quickly than by using conventional methods. Our results suggest that this pyrF -based counterselection system can also be applied to other Streptomyces species and will greatly improve our ability to explore the potential of Streptomyces . 5 Conclusions 1 Streptomyces produces a remarkably diverse array of secondary metabolites, including antibiotics, antitumor compounds, immunosuppressants, and antivirals. However, targeted gene disruption can be inefficient in industrial Streptomyces strains due to the lack of suitable selectable markers and a low frequency of HR. Here, we have demonstrated the feasibility and advantage of using the pyrF gene as a counterselectable marker to create marker-free mutants of S. rimosus . This pyrF -based counterselectable system was very effective and enabled us to obtain the target gene knockout mutants by analyzing a small number of clones, and the time to obtain the desired mutants was greatly shortened. 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Mol Ther Nucleic Acids , 4 : E264 . https://doi.org/10.1038/mtna.2015.37 https://doi.org/10.1038/mtna.2015.37 Off-target effects in CRISPR/Cas9-mediated genome engineering Zhao YW , Li L , Zheng GS , et al. , 2018 . CRISPR/dCas9-mediated multiplex gene repression in Streptomyces . Biotechnol J , 13 ( 9 ): 1800121 . https://doi.org/10.1002/biot.201800121 https://doi.org/10.1002/biot.201800121 CRISPR/dCas9-mediated multiplex gene repression in Streptomyces Yiying YANG, Qingqing SUN, and Yang LIU were responsible for the methodology, data curation, and formal analysis. Hanzhi YIN, Wenping YANG, and Yang WANG contributed to strain resources and supervision. Ying LIU and Yuxian LI contributed to the software and sequence alignment. Shen PANG, Wenxi LIU, Qian ZHANG, Fang YUAN, and Shiwen QIU contributed to the strain fermentation and composition analysis by HPLC. Jiong LI and Xuefeng WANG performed the fermentation experiments. Keqiang FAN, Weishan WANG, Zilong LI, and Shouliang YIN contributed to the investigation, project administration, supervision, writing, review, and editing. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work is supported by the Natural Science Foundation of Hebei Province (No. C2019209399), Tangshan Science and Technology Project (No. 20130208b), the Science and Technology Program of Hebei (No. 18222916), and the Research Fund for Top Discipline Construction of North China University of Science and Technology (No. 18060720), China. We wish to thank Professor Meijin GUO (East China University of Science and Technology, Shanghai, China) for providing Streptomyces rimosus M4018. We dedicate this article to the memory of our friend and mentor Keqian YANG (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), who made important contributions to understanding the biosynthesis and function of secondary metabolites in Streptomyces. 10.1631/jzus.B2000606 1673-1581（2021）05-0383-14 2020-12-23 2021-05-15 2022-05-09 © Zhejiang University Press 2021 Yiying YANG, Qingqing SUN, Yang LIU, Hanzhi YIN, Wenping YANG, Yang WANG, Ying LIU, Yuxian LI, Shen PANG, Wenxi LIU, Qian ZHANG, Fang YUAN, Shiwen QIU, Jiong LI, Xuefeng WANG, Keqiang FAN, Weishan WANG, Zilong LI, and Shouliang YIN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 22