A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora

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A CRISPR-Cas9 strategy for activating the Saccharopolyspora erythraea erythromycin biosynthetic gene cluster with knock-in bidirectional promoters Yong Liu, Chong-Yang Ren, Wen-Ping Wei, Di You, Bin-Cheng Yin, and Bang-Ce Ye ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00024 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019

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ACS Synthetic Biology

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A CRISPR-Cas9 strategy for activating the Saccharopolyspora erythraea

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erythromycin biosynthetic gene cluster with knock-in bidirectional promoters

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Yong Liu a, Chong-Yang Ren b, Wen-Ping Wei a, Di You a, Bin-Cheng Yin a, and Bang-Ce Ye a,b,c*

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[Author affiliations]

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aLaboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. bInstitute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River

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Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China. cSchool of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China.

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Yong Liu. E-mail: [email protected]

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Chong-yang Ren. E-mail: [email protected]

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Wen-Ping Wei. E-mail: [email protected]

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Di You. E-mail: [email protected]

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Bin-Cheng Yin. E-mail: [email protected]

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[Corresponding author]

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*To whom correspondence should be addressed, E-mail: [email protected]

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ACS Paragon Plus Environment

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ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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The regulation of biosynthetic pathways is a universal strategy for industrial strains that

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overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive

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clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were

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tested by reporter mCherry. The SACE_0720 (eryBIV) - SACE_0721 (eryAI) spacer was selected

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as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs)

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were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-

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Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native

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promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression

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increased 32 and 79 times, respectively. High performance liquid chromatography results showed

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that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in

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bidirectional promoter knock-in recombinant strains. Based on the activated strain Ab::Pj23119-

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PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected

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erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize

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the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study

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showed that bidirectional promoter knock-in and CRISPR interference could regulate gene

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expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster

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activation and gene regulation in Actinobacteria.

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Keywords: CRISPR-Cas9, Bidirectional promoters, BGC activation, Saccharopolyspora

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erythraea, Over-expression, CRISPRi

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Abbreviations1

6dEB, 6-deoxyerythronolide B; BAC, bacterial artificial chromosome; BDP, Bidirectional promoter; BGC, biosynthetic gene clusters; CATCH, Cas9-assisted targeting of chromosome segments; CRISPRi, clustered regularly interspaced short palindromic repeats interference; GABA, gamma-aminobutyric acid; NP, natural product; NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; TSB, tryptic soy broth 1


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Microbial natural products (NPs) have proven to be a major storehouse for developing bioactive

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chemical compounds in recent decades.1, 2 Natural products from the secondary metabolism of

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Actinomycetes, in particular, have important pharmaceutical applications, which include use in

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medicine for the treatment of cancers, and antibiotics for treatment of infections.3-5 The genes

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encoding the biosynthetic pathways responsible for the production of secondary metabolites are

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usually clustered together on the chromosome in biosynthetic gene clusters (BGCs),6 which range

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in size from several tens to hundreds of kilobases.7 Genomic insight or mining of such BGCs can

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improve their potential ability to produce new antibiotics and other natural pharmaceuticals,8 and

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has accelerated the understanding of their molecular biosynthetic mechanisms.2, 9 BGCs are often

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composed of a backbone gene, polyketide synthases (PKSs), and non-ribosomal peptide

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synthetases (NRPSs); these enable the generation of a chemical back-bone structure and its

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subsequent modification.10 For example, the macrocyclic aglycone of erythromycin, 6-

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deoxyerythronolide B (6dEB), was synthesized by 6-deoxyerythronolide B synthase (DEBS).11

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Polyketide synthase (PKS) encoding genes include SACE_0721 (eryAI), SACE_0723 (eryAII),

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SACE_0724 (eryAIII).9, 12

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing has

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been successfully applied in several Gram-positive bacteria, which to date have included B.

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subtilis,13, 14 M. tuberculosis,15, 16 M. smegmatis,17 S. coelicolor,18-20 and S. erythraea.21

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Researchers applied CRISPR-Cas9 to precisely and efficiently introduce heterologous strong

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promoters into hosts for BGC activation.2, 21, 22 Zhao et al. demonstrated knock-in that constitutive

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promoters replacing upstream promoter regions of main biosynthetic operons or pathway-specific

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activators triggered novel type II polyketide production and activated multiple BGCs of different

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classes in five Streptomyces species.22 Cas9-assisted targeting of chromosome segments

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(CATCH) has been used in vitro with Gibson23 assembly to clone large-sized BGC into bacterial

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artificial chromosome (BAC) vectors.24, 25 RecET recombineering system can markerless

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integration of BGCs for polyketide and isoprenoid into the P. putida chromosome.26

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Promoter engineering is a powerful synthetic biology tool for activating transcriptionally silent

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natural product BGCs,27, 28 and a well-characterized strong promoter is often preferable to the

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natural BGC promoter.29, 30 Bidirectional promoters (BDPs) allow for efficient co-expression of

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genes to activate BGC.31 CRISPR interference (CRISPRi) constitutes a powerful strategy for

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regulating gene expression at the transcriptional level in synthetic biology.32-34 CRISPRi has been

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applied in metabolic engineering to increase the yields of targeted biochemicals.32 In E. coli, for

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example, it has been used to improve production of P(3HB-co-4HB),35 β-carotene,36 and

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butanol,37, 38 and to increase the rate of xylose utilization.39 Researchers have also applied the


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CRISPR-Cas9 system for genome engineering of gamma-aminobutyric acid (GABA) production

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in C. glutamicum.40 A further example is that of green light responsive transcriptional factor,

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CarH, which regulates the expression of the carotenogenic gene cluster.41 Its green light

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sensitivity and activity is dependent on AdoB12 (5’deoxyadenosylcobalamin), which has been

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used in synthetic biology as an inducible gene switch.33

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In this study, we used the CRISPR-Cas9 strategy to knock-in BDPs at the spacer between

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SACE_0720 and SACE_0721 in order to activate the erythromycin BGC in S. erythraea. Based

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on the activated strain Ab::Pj23119-PkasO, aceA and sdhA interference affected growth. In

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summary, this study showed that BDP knock-in and CRISPRi can regulate gene expression in S.

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erythraea. This strategy has potential application in biosynthetic gene cluster activation and gene

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regulation in Actinobacteria.

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Results and Discussion

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Analysis of the Erythromycin BGC Promoters and Combination of BDPs

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First, we examined whether the spacer sequence in the erythromycin synthesis gene cluster

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(Figure S1) contain promoters by cloning the spacer sequence and fusing it upstream of Flag-

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mCherry. Promoter strength was expressed by the reporter gene mCherry (Figure 1a, Figure 1c,

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Figure S2, Figure S3). All ten promoters were tested, with fluorescence intensity correlating to

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differences in promoter strength. The results showed that the promoter of SACE_0733 (PermE)

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was the strongest promoter from 24 h to 96 h, but that promoter of SACE_0720,

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SACE_0721(PeryAB) continued to increase past this timepoint. At the 96 h time point, the

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intensities of SACE_0720 and SACE_0721 were already greater than that of PermE (Figure 1a).

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In addition, we compared 0720P and 0721P with Pj23119, PkasO, with the results showing that

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Pj23119, PkasO is much stronger than the promoter of SACE_0720, SACE_0721 (Figure 1a and

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1b). The strength of each promoter was evaluated by quantifying the fluorescence intensity

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accumulated over 96 h of culture (Figure 1b). Of the three promoters tested, Pj23119 was the

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strongest, while PermE the weakest (Figure 1b).


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Figure 1 Characterization of bidirectional promoters for the erythromycin BGC

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(a) Flag-mCherry was driven by spacers (promoters) in S. erythraea. Fluorescence intensity per OD for

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Next, we used sfGFP and mCherry as reporters to verify the combined constitutive BDPs.

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Pj23119 and PkasO were artificially combined into a BDP (PkasO-Pj23119) (Figure S4), and two

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fluorescent proteins (sfGFP, mCherry) were used to verify whether the BDP could drive gene

M::Promoters-mCherry. (b) Characterization of promoters strength using RFU/OD. Comparison of SACE_0720 - SACE_0721 spacer and promoter (PermE, Pj23119, PkasO) strength. Fluorescence intensity/OD for M::0720P-mCherry, M::0721P-mCherry, M::PermE-mCherry, M::PkasO-mCherry, and M::Pj23119-mCherry. (c) Sequencing of Promoter (Spacer)-mCherry. Flag-mCherry was driven by 0720P (SACE_0720 Spacer), 0721P (SACE_0721 Spacer), and promoters (PermE, Pj23119, PkasO) in S. erythraea. (d) Schematic diagram of the construction of bidirectional promoters. Verification of bidirectional promoters by dual reporter sfGFP and mCherry. The sfGFP was driven by PkasO, and mCherry was driven by Pj23119. (e) Fluorescence intensity of the M::sfGFP-Pj23119-PkasO-mCherry. (f) Quantification of sfGFP of the recombinant strain (M::sfGFP-Pj23119-PkasO-mCherry) by RFU/OD at 24, 48, 72, 96 h. (g) Quantification of mCherry of the recombinant strain (M::sfGFP-Pj23119-PkasO-mCherry) by RFU/OD at 24, 48, 72, 96 h. For sfGFP, excitation was measured at 475 nm, and emission was measured from 495 nm to 750 nm. Sensitivity: 100%. For mCherry, excitation was measured at 575 nm, and emission was measured from 595 nm to 750 nm. Sensitivity: 100%.


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expression in both directions (Figure 1d). Following transformation of the recombinant plasmid

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into S. erythraea, the results showed that M::sfGFP-PkasO-Pj23119-mCherry has an absorption

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peak at 510 nm when excited at 475 nm, and another absorption peak at 610 nm when excited

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with 575 nm (Figure 1e, Figure S5), indicating that sfGFP and mCherry were successfully

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expressed. We selected four time points to characterize the expression trends of the reporter

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genes, and found that fluorescence of sfGFP and mCherry intensity at OD600 was consistent at

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various time points (Figure 1f and 1g, Figure S5).

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CRISPR-Cas9 Strategy for BDP Knock-in

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Through the previous comparative experiments, we determined the strength of each promoter,

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which were: Pj23119 > PkasO > PermE. In addition, considering that SACE_0721 (encoding

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eryAI, 6-deoxyerythronolide-B synthase) is the most important enzyme involved in erythromycin

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synthesis, we selected the spacer of SACE_0720 and SACE_0721 as the knock-in target (Figure

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2a, Figure S6-S9), and designed dual sgRNA for SACE_0720 - SACE_0721 spacer target

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sequences (Table S2). This allowed us to activate the whole erythromycin gene cluster for the

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production of erythromycin (Figure 2a). The plasmid was constructed according to the method

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described previously, and its sequence was confirmed (Figure S7, S9, and S10).

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We designed three types of BDPs: PermE-PkasO (Figure 2b), PkasO-Pj23119 (Figure 2d) and

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Pj23119-PkasO (Figure 2f). The recombinant strains featuring bidirectional promoter knock-in

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were selected and evaluated by PCR (Figure 2c, 2e, 2g). To ensure that the PCR product was

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amplified from the chromosome rather than the plasmid, primers were designed to anneal slightly

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upstream (outside of the homology arms 243 nt) and downstream (162 nt) of the editing template

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sequence (primers: KI-test-F, KI-test-R) (Figure 2 and Figure S7, S9). Each PCR product was

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sequenced with internal primers to determine if the knock-in had been introduced (Supplementary

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data).


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Figure 2 Bidirectional promoters knock-in through CRISPR-Cas9 strategy

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(a) Schematic representation of using CRISPR-Cas9 for introduction of BDP cassettes. Map of pKECas9-

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We next investigated the behaviors of BDP knock-in stains. We first analyzed their growth

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phenotype in TSB medium. Growth curves showed that bidirectional promoter knock-ins had no

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significant effect on their growth (Figure 3a). We then analyzed the recombinant strains by

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fermentation in industrial medium, with the Ab prior to transformation used as a control. The

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fermentation results showed that the yield of the recombinant strain Ab::Pj23119-PkasO was

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higher than that of the control from the fifth day onwards (Figure 3b and Figure S11, S12),

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reaching its maximum on day 8. From the yield curve, the recombinant strain Ab::Pj23119-PkasO

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was found to have an improved erythromycin production capacity within the timeframe of study

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(Figure 3b). Next, we determined the yield of Ab::Pj23119-PkasO using fermentation medium

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(Supplementary Methods), and found its yield to be increased by 58.3% (Figure 6) compared with

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the starting strain (Ab as control).

sgRNAII-UHA-P1P2-DHA. The expression of the Cas9 coding sequence is driven by the PermE promoter, and the expression of the dual sgRNAs cassette is under the control of the Pj23119 and PkasO promoters. Apramycin serves as the selection marker (Figure S6, S8) and can shuttle between E. coli and Actinomycetes. The three types of BDP knock-in are PermE-PkasO, PkasO-Pj23119, and Pj23119-PkasO. (b) and (c) PCR-based identification of recombinant strains using KI072-07021 test primer pairs (KI2021test-F and KI2021-test-R) and promoter primer pairs (For Ab::PermE-PkasO, PCR products with KI-F + kasO-R were 1813 bp, with ermE-F + KI-R were 1525 bp). (d) and (e) For Ab::PkasO-Pj23119, PCR products with KI-F + rk-j-R were 1662 bp, with rk-j-F + KI-R were 1374 bp. (f) and (g) For Pj23119PkasO, PCR products with KI-F + rj-k-R were 1662 bp, with rj-k-F + KI-R were 1374 bp.


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Figure 3 Growth curve and Erythromycin production for recombinant strains

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(a) Growth curve for the Ab (control), Ab::PermE-PkasO, Ab::PkasO-Pj23119, Ab::Pj23119-PkasO. (b)

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BDP Knock-in Activated the Erythromycin BGC

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With CRISPR-Cas9, we have demonstrated that efficient and precise introduction of BDPs drives

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expression of the erythromycin BGC and triggers the production of unique metabolites. We

Time course profile of erythromycin production (Supplementary Methods, bioactivity assay) in Ab::PermE-PkasO, Ab::Pj23119-PkasO, Ab::PkasO-Pj23119 grown in ABPM8 fermentation medium within the timeframe of study.

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compared the fermentation of three recombinant strains in industrial fermentation medium. The

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culture was sampled and RNA was extracted on the fourth day, after which the transcription level

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of erythromycin biosynthetic gene clusters was determined. The cultured product was collected

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until the 8th day. The results showed that the erythromycin BGC was activated in Ab::PermE-

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PkasO. Analysis of transcript levels showed that expression of the right side BGC of the target is

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generally higher than that of the left side (Figure 4a), which is consistent with the previously

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validated constitutive promoter strength (PermE < PkasO).


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The high performance liquid chromatography (HPLC) results revealed the presence of 3 peaks in

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the samples (dissolved in ethanol). Metabolites (3.925, 5.925, 6.575,18.925 min) were not

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detected in Ab (the parent strain used as control) (Figure 4d and Figure S11). The HPLC results

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show that the Erythromycin A peak disappears and a peak appears at the 7.9 min position (Figure

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4e). For Ab::Pj23119-PkasO, the entire gene cluster was successfully activated and Erythromycin

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A was significantly improved (the main peak of Erythromycin A at 10.166 min) (Figure 4c and

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4f), and have a peak appears at the 5.933 min position (Figure 4f). In the case of Ab::PkasO-

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Pj23119, only the left side of the BDP was activated successfully, and it is possible that the right

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side may have been lost as a result of the reorganization and repair process. The PCR verification

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results are presented in Figure 4b.

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Figure 4 qPCR and HPLC Determined the Activation of the erythromycin BGC

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(a)-(c) Transcriptional analysis of erythromycin BGC expression of Ab::PermE-PkasO, Ab::PkasO-

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The position of the erythromycin BGC on the genome is from 778654 (SACE_0713, eryK) to

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832825 nt (SACE_0734, eryCI). The size of the erythromycin BGC is 54171 bp (Figure 5a). To

Pj23119, Ab::Pj23119-PkasO at 4 d in ABPM8 medium. Relative transcript levels were obtained individually after normalization to the sigA internal reference gene. Gene expression values observed in the control strain (Ab) were set as 1.0. Error bars indicate the standard deviations from three independent replicates. (d)-(f) HPLC analysis of ethanol extracts from recombinant strains in which bidirectional promoters PermE-PkasO, PkasO-Pj23119 and Pj23119-PkasO were knocked into the spacer of SACE_0720 and SACE_0721.


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further confirm the erythromycin BGC activation, we selected Ab::Pj23119-PkasO cultured in

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TBS medium and analyzed its transcription level. Amplification curve reflects the difference

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between the internal reference (sigA) and SACE_0720 (eryBIV), SACE_0721(eryAI) (Figure 5c).

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The transcriptional results indicated that the entire erythromycin BGC was also successfully

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activated in TBS medium at 48 h time point. The transcript level of SACE_0720 (eryBIV) was

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increased 32 times, and SACE_0721 (eryAI) was increased 79 times (Figure 5b and 5c).

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Figure 5 Transcriptional analysis of Ab::Pj23119-PkasO in TSB medium

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(a) Schematic representation of erythromycin biosynthetic gene cluster and the Pj23119-PkasO knock in at

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Optimized the Metabolic Flow to Increase Erythromycin Production

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The CRISPRi system was successfully established as a technique for S. erythraea gene

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regulation, and continued the CRISPRi trial in a recombinant erythromycin-producing strain

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(Figure S13 and Figure S14). The methylmalonyl-CoA mutase (MutAB, SACE_5638 and

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SACE_5639) catalyzes the reaction is: (R)-methylmalonyl-CoA = succinyl-CoA. This reaction

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converting the citric acid cycle (TCA) intermediate succinyl-CoA and (R)-methylmalonyl-CoA.42

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Downregulation of sdh restricted the carbon metabolism entry into the TCA in erythromycin

SACE_0720-SACE_0721Spacer. (b) qRT-PCR analysis of erythromycin BGC expression at 48 h in TSB medium. Relative transcript levels were obtained individually after normalization to the sigA (SACE_1801) internal reference gene. (c) Amplification curve and melt peak curve for the Ab (Control) and Ab::Pj23119PkasO. In panels the cure showed sigA (SACE_1801), eryBIV (SACE_0720), and eryAI (SACE_0721).


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high-producing strain.43 The succinate dehydrogenase (sdhA and sdhB) with CRISPRi for

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regulating carbon flux to increase 4-hydroxybutyrate biosynthesis.35 We consider increasing

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succinyl-CoA to increase the pool of (R)-methylmalonyl-CoA. Two genes, namely sdhA

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(SACE_1170 and SACE_6584, encoding succinate dehydrogenase) and aceA (SACE_1149,

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encoding isocitrate lyase) (Figure S15) were selected to investigate the effects of their different

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expression levels on erythromycin A production (supplemental material). Considering that

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sgRNAs were able to bind to their respective targets with different efficiencies of repression due

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to their different specific targeting locations, we designed several sgRNAs. For sdhA, we

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compared template- and non-template-targeting sgRNA sequences designed to induce different

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levels of repression. When the first generation of recombinant strains was screened, erythromycin

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production increased slightly (Figure S16, S17). After the first passage, a fermentation

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experiment was carried out in a shake flask, and the biomass and erythromycin production were

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measured at different time points.

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Real-time qPCR results indicated that the CRISPRi system repressed target genes at the mRNA

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level. Interference affecting aceA expression affected the growth of the recombinant strains

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(Ab::PP1449iNT). Simultaneously, erythromycin production was also decreased (Figure S16).

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Similarly, when sdhA was silenced, it showed a similar tendency. In addition, culture growth as

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determined from OD600 results showed that CRISPRi recombinant strains silencing of target

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genes on the non-template strand had a more significant effect on growth (Figure S16d). Finally,

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the qPCR results showed the aceA transcript level was reduced (Figure S16c), and the sdhA

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transcript level was reduced in both template strand and non-template strand targeting (Figure

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S16f). Shake flask studies were carried out to investigate whether erythromycin production was

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affected aceA and sdhA were repressed. Compared with the control group Ab::PP, which lacked

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the CRISPRi system, the OD600 of Ab::PP6584i was lower (Figure S16d) and accumulated

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erythromycin A was decreased (Figure S16e). The sgRNA targets studied (for sdhA) were shown

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to be effective in reducing gene expression at different levels (Figure S15).


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Figure 6 Erythromycin production in different S. erythraea engineered strains

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Effect of erythromycin production when BDP (Pj23119-PkasO) knock-in and further engineered strains.

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When the amount of Apr was reduced by half and the culture temperature was raised to 37 °C, the

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effect of CRISPRi was found to be decreased. Growth inhibition of the strains decreased (Figure

Ab::PP indicated Ab::Pj23119-PkasO. T, indicated template strand; NT, indicated non-template strand. OE, over expression. 3398-3400, SACE_3398 to SACE_3400, propionyl-CoA carboxylase, pccB; 7038-7039, SACE_7038 and SACE_7039, propionyl-CoA carboxylase, accD1. Strains grown in fermentation medium for 6 d. HPLC analysis of ethanol extracts from recombinant strains.

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S17a and S17b, compared with Figure S17e and S17f), and the yield of Ab::PP6584iNT increased

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by 15.1% compared with Ab::PP (Figure 6).

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Bidirectional promoters (BDPs) offer the possibility of dramatically improving pathway design.

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BDPs allow for co-expression of genes on both strands in a single cloning.31 Furthermore, BDP

14

knock-in is an efficient method for activating BGCs, and its use has been demonstrated in the

15

discovery of previously known products in unknown producers, and of derivatives of new

16

compounds that are of potential pharmaceutical interest.22 In this study, we knocked-in BDPs at

17

the spacer of SACE_0720 and SACE_0721 using the CRISPR-Cas9 strategy, and successfully

18

activated the erythromycin BGC (Figure 4f). The recombinant strain Ab::Pj23119-PkasO was

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screened for increased erythromycin yield (Figure 3b and Figure 4c, 4f) resulting from activation


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of the entire BGC (both strands of the knock-in target). In addition, due to knock-in of different

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BDPs, the erythromycin BGC is partially activated, resulting in the production of new

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compounds. For Ab::PermE-PkasO, The HPLC results indicate the presence of metabolites in the

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product (Figure 4d), which were not detected in control strain Ab (Figure S11). Ab::PkasO-

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Pj23119 only activated the left side (eryK, eryBVII, eryBVII, eryCIV, eryBVI, eryCVI, eryBV,

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eryBIV ) of the erythromycin BGC (Figure 4b, 4e). These results indicated that the erythromycin

7

BGC had been activated. This work demonstrated that high-yield strains can be obtained using

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CRISPR-Cas9-mediated knock-in of BDPs in combination with high-throughput screening

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strategies.

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Overexpression of some genes in erythromycin BGC (eryK and eryG) have improved purity and

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production of erythromycin A.44 SACE_0721 (eryAI) is a critical enzyme in the synthesis of

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erythromycin. PeryAB (the spacer between SACE_0720 and SACE_0721) was previously used

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as a bidirectional promoter to drive the spinosyn BGC.45 Further qPCR analysis of the

14

erythromycin BGC results showed that the transcript level of SACE_0720 (eryBIV) was increased

15

by 32 times, and that the transcription level of SACE_0721 (eryAI) was increased by more than

16

79 times (Figure 5b) in Ab::Pj23119-PkasO strain in TSB at the 48 h time point. Furthermore, the

17

transcription level of eryBIV was increased 105 times, and eryAI was increased 20 times (Figure

18

4c), when sampled in ABPM8 at day 4. In brief, the results from both groups showed that the

19

entire erythromycin BGC had been successfully activated, and that expression of other genes of

20

the erythromycin BGC had also been increased to varying degrees (Figure 5). However,

21

SACE_0722 and SACE_0729 did not improve because they are located on the complementary

22

strand, opposite to the genes from SACE_0721 to SACE_0732. This result agrees with previous

23

results which also showed that the transcription of SACE_0722 and SACE_0729 was not

24

increased.21

25

On the basis of the Ab::Pj23119-PkasO strain, we further overexpressed propionyl-CoA

26

carboxylase, pccB (SACE_3398 to SACE_3400), and accD1 (SACE_7038 and SACE_7039), but

27

found that this resulted in only slight increases in the yield of erythromycin A, which were not

28

statistically significant (Ab::PP/OE3398-3400, Ab::PP/OE7038-7039) (Figure 6). By further

29

optimizing metabolic flow with CRISPRi-based transcriptional control32, 46, 47 in order to increase

30

erythromycin A production, we achieved efficient simultaneous repression of two genes (aceA

31

and sdhA) involved in regulation growth and production of erythromycin A in recombinant

32

strains. When the first generation of recombinant strain were screened, erythromycin production

33

increased slightly (supplemental material). After the first passage, a fermentation experiment was

34

carried out in a shake flask, and the biomass and erythromycin production at different time points


13


Page 14 of 21

1

were measured, with the results showing that inhibition of aceA affects the growth of

2

recombinant strain (Figure S17a).

3

Previous research demonstrated that sgRNAs targeting the template (T) or non-template strands

4

(NT) of the promoter regions are very effective for gene repression. However, effective

5

repression is only observed on the NT strand when sgRNAs are targeted to the coding region.48, 49

6

We selected sdhA as a target gene to compare the inhibitory effects of the T and NT strands.

7

Moreover, because pKE-dCas9-sgRNA is a temperature-sensitive plasmid, we could control its

8

copy number by adjusting the temperature and controlling the dose of apramycin to achieve

9

different inhibition effects. The finding that optimized temperature control during fermentation

10

further increases erythromycin production may result in this control strategy becoming more

11

widely used in synthetic biology.

12

This study is the first to analyze erythromycin BGC activation through BDP knock-in in S.

13

erythraea. Regulation of the strength of BDPs, and inhibition of target genes, can optimize the

14

metabolic flux distribution and increase the yield of useful secondary metabolites. Our results

15

highlight the potential of this species as a source of new antibiotics and other pharmaceuticals.

16

Methods

17

Recombinant plasmid construction using the Hieff Clone Multi One Step Cloning Kit (Yeasen,

18

Shanghai, China). Plasmid isolation was performed using the Endofree Mini Plasmid Kit II

19

(Tinagen, Beijing, China). S. erythraea mutant strains were verified by colony polymerase chain

20

reaction (PCR). All plasmid constructs (Supplementary Material) were verified by sequencing

21

carried out using Phanta Max Supper-Fidelity DNA Polymerase (Vazyme, Nanjing China). All

22

enzymes and kits are listed in Table S1. Restriction enzymes, polymerases, and kits were used

23

according to the supplier’s instructions (Takara, Japan).

24

Microorganisms and Culture Medium

25

The recombinant strains used in this study are listed in Table S1. Escherichia coli DH5α was used

26

for construction of recombinant plasmids. Cells were cultivated on 90 mm disk using Luria

27

Bertani medium (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L) at 30 °C or 37 °C, flasks

28

with shaking at 220 rpm, supplemented with apramycin (100 μg/mL), as required, during plasmid

29

cloning. For selection of apramycin resistant S. erythraea mutant strains after transformation,

30

either 25 μg/mL or 50 μg/mL apramycin was used. The S. erythraea wild-type strain

31

(NRRL23338) and the erythromycin high producing strain (Ab) were grown on R2YE agar

32

plates.50 For seed-stock preparation, strains were cultured in 250 mL flasks containing 30 mL of


14

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

tryptic soy broth (TSB) for 48 h at 30 °C, with shaking at 220 rpm. Following this, 0.5 mL of

2

seed cultures were inoculated into a 500-mL flask containing 50 mL TSB medium, under the

3

same culture conditions, and the cell samples were harvested at the indicated time points (48 h)

4

for RNA extraction.

5

Construction of Promoter Test Plasmids

6

All pSET15251, 52 plasmids were assembled using a Hieff Clone Multi One Step Cloning Kit

7

(Yeasen, Shanghai, China). To construct the plasmid pSET152-promoter-mCherry, promoters

8

(spacers of SACE_0720-0721, SACE_0722-0723, SACE_0731-0732, SACE_0733-0734, Figure

9

S1) were amplified by PCR using S. erythraea genomic DNA as template, fused with mCherry by

10

overlap-extension PCR (Table S3), and cloned23 into a pSET152 (EcoRV, XbaI) backbone to

11

obtain the final plasmids (Table S4). Promoters PermE,53 Pj23119,54 and PkasO55, 56 were

12

maintained in our laboratory.21 Plasmids were confirmed by sequencing (Majorbio, Shanghai,

13

China), following which PEG-mediated transformations of protoplasts (S. erythraea WT and Ab)

14

were performed as previously described.52 Furthermore, we also constructed pSET152-sfGFP-

15

PkasO-Pj23119-mCherry.

16

Bidirectional Promoter Knock-in with CRISPR-Cas9

17

A Cas9 expression plasmid for S. erythraea, pKECas9, was previously constructed in our

18

laboratory.21 The sgRNAII expression and HA plasmid was constructed in three steps: (1) UHA

19

(SACE_0720) and DHA (SACE_0721) were amplified from S. erythraea genomic DNA. (2) a

20

SACE_0720-SACE_0721 spacer fragment with an NCC region complementing the PAM

21

sequence, or any 5′-NGG, is used as the target model. Dual sgRNA (mark as sgRNAII) targeting

22

SACE_0720-SACE_0721 spacer design followed previously described principles.21, 57 We

23

selected sgRNA sequences using Ape and used the BLAST function at the NCBI to search for

24

whole genome matches. Based on search results, the best (the number of matches on whole

25

genome is 1) sgRNA was selected. The resultant RNA expression cassette was then synthesized

26

in its entirety by Sangon Biotech (Shanghai, China, supplementary materials). (3) sgRNAII was

27

ligated upstream of UHA to generate H-sgRNAII-UHA, which was then fused with Pj23119-

28

PkasO and DHA through overlapping PCR. Finally, the whole cassette (H-sgRNAII- Pj23119-

29

PkasO-UHA-H) was cloned into the XbaI and HindIII sites of pKECas9 to generate pKECas9-

30

2021sgRNAII-UHA-Pj23119-PkasO-DHA for genomic modification of the SACE_0720-21

31

spacer in S. erythraea.58, 59

32

Correct plasmid assembly was confirmed by sequencing (Majorbio, Shanghai, China), and the

33

plasmids (listed in Table S1) were subsequently used to transform S. erythraea. Primers used for


15


Page 16 of 21

1

plasmid construction and cloning confirmation are listed in Table S2. The gRNA sequences and

2

PAM sequence used in sgRNA design for SACE_0720-SACE_0721 spacer deletions are listed in

3

Table S6.

4

The S. erythraea genome was extracted using a TIANamp Bacteria DNA kit (Tiangen, Beijing,

5

China). The Up-(UHA-Pj23119-PkasO-DHA)-Down long fragment was amplified from genomic

6

DNA using KI2021 test primer pairs (Table S2). PCR was performed using a T100 Thermal

7

Cycling Platform (Bio-Rad, USA) and Phanta Max Super-Fidelity DNA polymerase (Vazyme

8

Biotech, Nanjing, China) (Tables S3, S5). PCR products were confirmed by Sanger sequencing.

9

RNA Extraction, cDNA Synthesis and Quantitative Real-time PCR Analysis

10

Total RNA was isolated and purified from recombinant S. erythraea strains using an RNAprep

11

pure Cell/Bacteria Kit (Tiangen, Beijing, China). RNA quality was assessed using 1.2% agarose

12

gel electrophoresis, and then quantitative determined of concentration using a BioTek Reader. To

13

synthesize cDNA, 1.0 μg RNA was used with a PrimeScript RT Reagent Kit with gDNA Eraser

14

(Takara, Japan), with RNase-free water added to give a final concentration of 50 ng/μL. qRT-

15

PCR experiments were performed using a CFX96 Real-Time System (Bio-Rad, USA). Each 20.0

16

μL reaction contained 10.0 μL SYBR Premix Ex Taq GC (Takara, Japan). The reaction

17

parameters were: initial denaturation at 95 °C for 5 min, then 40 cycles (95 °C for 10 s, 60 °C for

18

20 s, and 72 °C for 30 s), with final extension at 72 °C for 10 min. A final dissociation step was

19

used to generate a melting curve in order to verify the specificity of the amplification procedure.

20

Real-time PCR was monitored and analyzed using CFX software (Bio-Rad, USA), and relative

21

expression levels were normalized to mRNA derived from sigA (SACE_1801) as the internal

22

standard.50 Levels of gene expression are shown as fold-change relative to the time point 1

23

exponential growth phase sample. All samples were prepared in triplicate to obtain the Ct values,

24

and the relative level of gene expression for each mutant (compared with Ab) was calculated

25

using the comparative Ct method (2-△△Ct).21, 60 All primers used in this work are described in

26

Table S2.

27

Biomass and Evaluation of Secondary Metabolites

28

The S. erythraea high erythromycin producing strain (Ab, control) and its mutants were cultured

29

in triplicate in 30 mL of TSB at 30 °C. Growth was analyzed using a microplate reader (BioTek

30

Reader). Cell density measurements at OD600 were acquired every 12 h. Data were analyzed using

31

GraphPad Prism 7 software package (GraphPad Software).21 To evaluate erythromycin

32

production, the production yield of the engineered strains (0.5 mL of the TSB preculture) was


16

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

assessed in fermentation medium. Laboratory scale fermentations were performed in liquid

2

culture at the 50 mL scale in 500 mL flasks at 30 °C and 250 rpm for nine days.43 For analysis of

3

secondary metabolites produced by bidirectional constitutive promoter knock-in recombinant

4

strains, samples (freeze-dried powders from fermentation supernatants were dissolved in ethanol,

5

and the solutions were passed through a 0.22 µm filter) were separated on an LC-2030 system

6

(Shimadzu) using a Diamonsil Plus 5 μm C18-A, 250 × 4.6 mm column (DiKMA), using a linear

7

gradient of solvent B (55% acetonitrile) against solvent A (1 L water with 8.7 g K2HPO4, pH 8.2)

8

at a flow rate of 1.0 mL/min and with the column maintained at 40 °C. UV spectra were acquired

9

at 215 nm.21 The fermentation products of TSB and ABPM8 medium was firstly evaluated with

10

the bioactivity assay to determine the titer of erythromycin (Supplementary Methods).

11

Quantitative Measurement of Fluorescence

12

For S. erythraea, 1 mL of the 24-h seed cultures of the recombinant strains was inoculated into 30

13

mL of TSB medium and cultured for 24, 48, 72, and 96 h in 500 mL flasks at 30 °C and 220 rpm.

14

Aliquots (1 mL) of the cultures were washed twice with PBS (137 mmol/L NaCl, 2.7 mmol/L

15

KCl, 10.1 mmol/L Na2HPO4, 1.8 mmol/L KH2PO4, pH 7.2) and resuspended in 1 mL of PBS,

16

after which sfGFP and mCherry fluorescence was detected using a BioTek Reader (for sfGFP,

17

excitation at 475 nm and emission at 510 nm; for mCherry, excitation at 575 nm and emission at

18

610 nm). All fluorescence values were normalized to cell growth (OD600) for 24, 48, and 72 h,

19

respectively. Values and error bars represent the average and standard deviation of three

20

experimental replicates, respectively.

21

CRISPR interference

22

Plasmid pKE-dCas9 (Figure S10, S11) was constructed based on pKECas9,21 which contains a

23

gene encoding the dCas9 (D10A and H840A functionally inactivate the RuvC and HNH

24

domains) protein.61 To prepare the pKE-dCas9-sgRNA plasmids for gene interference, the target

25

sgRNA used a homology arm ligated to the XbaI and HindIII sites of pKE-dCas9. An sgRNA for

26

S. erythraea contains three parts: a Pj23119 promoter, a 20 bp DNA region complementing the

27

gene sequence of interest, and an 82 bp hairpin region (for dCas9 protein binding) with a

28

terminator. The 20 bp sequence complementary to the target site was designed with Ape (Tables

29

S7, S8, and S9), which selected efficient target sgRNAs.48, 62 The forward and reverse primers (H-

30

sgRNA-F, H-sgRNA-R) were subsequently annealed to obtain a double-stranded inserted

31

fragment precisely fitting the pKE-dCas9 vector. This method allows for convenient changes in

32

the complementary region to suit any interesting gene. Recombinant strains with CRISPRi were


17


1

grown in TSB in triplicate 30 mL cultures. Apramycin (100 μg/mL) was added when cultured at

2

30 °C, but at 37 °C, apramycin (25 μg/mL) was added. Plasmids used in the construction of the

3

CRISPRi platform suitable for S. erythraea are listed in Table S1. Primers used for site mutation

4

of dCas9 are listed in Table S1. Table S2 lists all primers used in plasmid construction and tests.

5

Supporting Information

6

Supporting Methods, Figures S1−S17, and Tables S1−S9. Erythromycin biosynthetic gene

7

cluster; Plasmids construction; Fluorescence and OD600 measurement; Erythromycin bioactivity

8

assay; HPLC analysis; Identification of BDP knock-in strains; Site-point mutation of Cas9 to

9

dCas9; CRISPRi trial; Primers and sgRNAs sequences.

Page 18 of 21

10

Authors' contributions

11

Yong Liu, was responsible for experimental design, investigation, analysis, interpretation of data,

12

and writing the original draft. Chong-Yang Ren, participated in HPLC and Growth curve data

13

acquisition. Wen-Ping Wei analyzed HPLC data. Di You and Bin-Cheng Yin provided advice and

14

supervised the research. Bang-Ce Ye was responsible for the study’s conception and design, data

15

analysis, and final approval of the manuscript. All authors read and approved the final

16

manuscript.

17

Acknowledgements

18

This work was supported by grants from the National Natural Science foundation of China

19

(31730004 and 21575089).

20

Declarations of interest: none

21

References

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora

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