A flexible , modular and versatile part assembly toolkit for gene cluster engineering in Streptomyces

Izhodiščni članek: [1]

Introduction

One of the most used species of bacteria in the industry is Streptomyces, it has found use in the production of natural products such as immunosuppressants, antibiotics, herbicides and antitumor drugs. With the recent development in genome sequencing, a large unused pool of gene clusters(BGCs) was discovered in Streptomyces, which have the potential to lead to new bioactive compounds. So far attempts to activate said BGCs have been made using cloning and refactoring, heterologous expression in order to overcome limitations posed by individual activation methods. This approach is sadly limited by the lack of standard and versatile toolkits and assembly technologies. Despite the fact that genetic engineering has been used to boost the production of secondary metabolites in Streptomyces for decades using vector platforms (such as pIJ family), these vectors systems are mainly limited to single genes and they also are incompatible with advanced standards such as Biobrick and Golden Gate. In order to fix all of these issues, they decided to design a flexible and modular DNA assembly strategy that allows easy exchange of plasmid copy numbers, selection marker genes, integration sites, regulatory and catalytic parts of gene clusters. Furthermore it enables cloning and editing of different-sized gene clusters using various cloning methods such as CATCH (Cas9-Assisted Targeting of CHromosome segments) and yeast homologous recombination based DNA assembly.

Materials and methods

Throughout this study they used Escherichia coli EPI300 for molecular cloning and plasmid propagation and E.coli ET12567/pUZ8002 was used for E. coli-Streptomyces conjugation. The strains were grown at 37 °C in LB medium (10g/L tryptone, 5g/L yeast extract and 10g/L NaCl) with the corresponding antibiotic. At the same time they also used S. coelicolor, S. albus and S/ lividans strains which were cultures on MS medium (20g/L soybean flour, 20g/L mannitol and 20g/L agar) which were used for sporulation and conjugation. S. venezuelae was used for the same purpose and grown in MYM medium (4.2 g/L (D-(+)-maltose monohydrate, 4 g/L yeast extract, 4 g/L malt extract, and 20 g/L agar). The antibiotics they used were ampicillin, thiostrepton, apramycin, hygromycin, spectinomycin.

They introduced the vectors into four Streptomyces species by conjugation using standard procedure and selected exconjugants on MS or MYM agar with nalidixic acid and the appropriate antibiotic.

They used two plasmids pTHS-XGSN* and pPAS-PT as basic vectors to carry the T7 RNA polymerase and T7 promoters. To boost the expression of T7 RNAP they replaced four rare TTA leu codonds with CTC leu. The T7 RNAP* fragment amplified with T7 RNAP*-F and T7 RNAP*-R primers was cloned using the Golden Gate method into pTHS-XGSN*. Using OLMA (Oligonucleotide Linkers-Mediated Assemble) they constructed the pPAS-PT7(x) series plasmids in their lab. They inserted the pTHS-T7 RNAP* cassette into the chromosome of S. albus J1074 and in the resulting strains pPAS-PT7 was introduced which carries the T7 promoters. The strains were grown in TSB medium and after adding cumate to the medium the expression of T7 RNAP* would be activated. As a positive control they used a kasOp* promoter inserted into pPAP-PT to generate pPAS-kasOp* plasmid.

Results

To get around the problem of activating the silent gene clusters due to lack of the ability to manipulate multiple regulatory and multiple catalytic parts in a cross-species manner, they developed a modular and versatile DNA assembly toolkit for gene cluster engineering. All vectors in the toolkit include a cargo part(part 1) and an essential part(part 2). The cargo part is composed of the cloning sites of the regulatory and catalytic parts while the essential part is responsible for the replication origins of the plasmid, selection marker genes in E. coli, yeast and Streptomyces.

The cargo part has two transcription terminators T1 and T2 and also contains a multiple cloning site (MCS). The MCS was designed in a specific way to allow compatibility with standard cloning system. It was designed as follows: I-SceI–EcoRI–XbaI–BsaI-BsaI–SpeI–PstI–I-SceI. EcoRI, SpeI, XbaI and PstI sites allow biobrick assembly, and two BsaI sites allow Golden Gate assembly, while the two homing endonucleases I – SceI sites are suitable for exchanging large fragments. The essential part is made up of four modules that are arranged in the same order in all vector: 1- Integration/replication module in Streptomyces: 2- Antibiotic markers; 3- Replication module in E. coli; 4- Replication module in yeast. As a result they constructed 10 basic vectors (shown below in figure 2). Large DNA gene clusters are more stable in a low-copy vector, so their toolkit includes: 1- pPAB and BAC replicons for large sized gene clusters >50kb 2- pPAS, pVHS and pTHS with pSC101 replicon for medium sized <50kb 3- pPAP, pVSP, pTSP and pIATP with p15A replicons for <30kb 4- pIATU with pUC replicon for small size gene clusters <10kb The vectors were conjugated from E. coli to the different strains of Streptomyces in order to test the conjugation frenquency. S. Lividans TK24 was found resistant to spectinomycin hence the plasmids with the spectinomycin antibiotic marker cannot be used. The toolkit is compatible with a wide range of DNA assembly approaches, BioBrick, Golden Gate, CRISPR/Cas9-based methods. A. Biobrick – by digesting the donor part with SpeI and EcoRI and the target vector by EcoRI and XbaI, they can be ligated using T4 DNA ligase and generate the recombinant plasmid. B. Golden Gate – PCR product or fragment is inserted in the donor vector flanked by specific four-base overhangs and BsaI resctriction site which can then be assembled into the vector backbone containing two BsaI restriction site. (also works with double stranded DNA oligos)

Controlled gene expression is necessary for improvement of secondary metabolite production and in order to facilitate gene expression optimization they developed a rapid, simple and standardized gene expression assembly vectors based on pTHS. Three of which are constituve (pTHS-XGe, pTHS-XGk, pTHS-XG23) and one is an inducible expression vector compatible with Golden Gate (pTHS-XGSN containing a tight cumate-inducible promoter PcymR*.

Actinorhodin (ACT) is a blue pigment produced by S. coelicolor M145 encoded by the act gene cluster. They cloned the act gene cluster (26kb) in pPAB vector using CATCH. The correct recombinant plasmids were verified by PCR and restriction enzyme digestion. It was predicted that the act gene cluster has five biosynthetic operons and two regulatory genes, so in order to improve the production of actinorhodin the network of the cluster was refactored by inserting well-characteried promoters P1-P2 to control actVA and actVI operons, P3 promoter for actII-orf2 and P4-P5 for actIII and actI operons. Editing the gene cluster was done by digesting the pPab-act with Cas9 and sgRNAs in vitro then they were co transformed into yeast together with DNA cassettes and homology arms matching each digestion site.

Conclusion

They made a toolkit that allows manipulation and optimization of large gene clusters and have proven it to be effective and successful to clone and refactor the act BGC, improving the production of actinorhodin. Using this toolkit they have provided an efficient promoter screening system that works for both assembly and testing of promoters, they hope that in the future more strains of actinomyces can be tested and expand the application of their toolkit.

Literatura

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