MODULATION OF GENE EXPRESSION

Abstract

The present invention provides methods for identifying a factor which modulates gene expression comprising providing a library of at least 110.sup.6 nucleic acid molecules each comprising a stochastic sequence of at least 50 nucleotides, introducing said nucleic acid molecules into nucleic acid constructs which comprise a reporter sequence and which do not comprise a further separate sequence upstream of the reporter sequence that can modulate expression of the reporter sequence to generate test constructs, introducing test constructs into host cells and assessing expression of the reporter sequence, thereby to identify a factor which modulates gene expression. Libraries of nucleic acid molecules, test constructs and host cells for use in such methods are also provided.

Claims

1. A method of identifying a factor which modulates gene expression, said method comprising: a. providing a library of at least 110.sup.6 nucleic acid molecules, wherein each nucleic acid molecule comprises a stochastic sequence of at least 50 nucleotides, optionally wherein each nucleic acid molecule also comprises an identical pre-determined and fixed sequence either adjacent to or within the stochastic sequence, wherein said pre-determined and fixed sequence does not include a promoter sequence itself capable of initiating transcription of a gene; b. introducing nucleic acid molecules from said nucleic acid library of (a) into nucleic acid constructs to prepare a library of test constructs, wherein each test construct comprises a nucleic acid molecule of part (a) upstream of a reporter sequence, and wherein each test construct does not comprise a further separate sequence upstream of the reporter sequence that can modulate expression of the reporter sequence; c. introducing test constructs from said test construct library of (b) into host cells to prepare a library of host cells, wherein host cells in the host cells library comprise a test construct; d. assessing expression of the reporter sequence in host cells from the library of host cells; and e. based on the level of expression of the reporter sequence, identifying a factor which modulates gene expression.

2. The method of claim 1, wherein to prepare the library of host cells in step (c), host cells are contacted with the test construct library of (b).

3. The method of claim 1 or claim 2, wherein in step (d) expression of the reporter sequence takes place under a test condition.

4. The method of any one of claims 1 to 3, wherein step (e) comprises identifying the nucleic acid molecule of the test construct from a host cell having a level of expression of the reporter sequence that is of interest and/or identifying a test condition under which a host cell expresses the reporter sequence at a level of interest, thereby to identify a factor which modulates gene expression.

5. The method of any one of claims 1 to 4, wherein the factor is a genetic element, a regulatory molecule present in the host cell, or a test condition.

6. The method of any one of claims 1 to 5, wherein the factor is a genetic element and the identifying of step (e) comprises sequencing the stochastic sequence.

7. The method of any one of claims 1 to 6, wherein the method is for identifying an element which modulates gene expression and wherein the element is a regulatory sequence which regulates gene expression, a binding site for a regulatory molecule, a binding site for an enzyme, a sequence which interacts with an expression-modulating factor, or a sequence responsive to a condition which modulates gene expression.

8. The method of claim 7, wherein the element is a transcriptional and/or translational control sequence, or a sequence which interacts with a transcription and/or translation modulating factor or a sequence which is transcribed into RNA and affects the stability and/or function of the RNA transcript.

9. The method of claim 7 or 8, wherein said element is a promoter, enhancer, silencer, operator region, a 5 UTR sequence, a ribosome binding site, a polymerase binding site, a binding site for an inducer or repressor of transcription, or a binding site for a transcription factor or any other factor which directly or indirectly modulates gene expression.

10. The method of any one of claims 1 to 9 wherein said method is a method of identifying an element which modulates gene expression under a test condition, wherein step (d) comprises: (i) incubating the library of host cells under a test condition; and (ii) assessing expression of the reporter sequence in host cells from the library of host cells; wherein the nucleic acid molecule identified in step (e) is an element which modulates gene expression under a test condition.

11. The method of any one of claims 1 to 4, wherein said method is a method of identifying a test condition which modulates gene expression, wherein step (d) comprises: (i) incubating the library of host cells under a test condition; and (ii) assessing expression of the reporter sequence in host cells from the library of host cells; wherein step (e) comprises identifying a nucleic acid molecule of the test construct from a host cell which expresses the reporter sequence under the test condition, and thereby identifying whether the test condition modulates gene expression.

12. A method of identifying a test condition which modulates gene expression, said method comprising performing steps (a)-(d) as defined in claim 1 or 2, wherein step (d) comprises: (i) incubating the library of host cells under a test condition; and (ii) assessing expression of the reporter sequence in host cells from the library of host cells; and wherein said method further comprises identifying whether the test condition modulates gene expression.

13. The method of any one of claims 1 to 12, wherein the stochastic sequence in each nucleic acid molecule comprises one or more biased random sequences.

14. The method of claim 13, wherein said one or more biased random sequences is A/T rich.

15. The method of claim 14, wherein each nucleic acid molecule comprises A/T rich sequences at the 35 and/or 10 regions.

16. The method of any one of claims 1 to 15, wherein the stochastic sequence in each nucleic acid molecule comprises an identical fixed ribosomal binding site or part thereof.

17. The method of claim 16, wherein said fixed sequence is a translation-modulating sequence, preferably a Shine-Dalgarno sequence or a Kozak sequence.

18. The method of any one of claims 10 to 17, wherein the test condition is selected from any one of temperature, pH, oxygen saturation and/or light, or the presence or absence of a molecule or added factor, e.g. a chemical factor.

19. The method of any one of claims 1 to 18, wherein the library of host cells is incubated in or on a growth medium supplemented with one or more chemicals.

20. The method of claim 19, wherein said chemical is a sugar, amino acid, peptide, protein, antibiotic, a putative regulatory compound, or a test candidate compound.

21. The method of any one of claims 6 to 20, wherein the element is for modulating gene expression in a prokaryote, preferably P. putida or E. coli.

22. The method of any one of claims 6 to 20, wherein the element is for modulating gene expression in a eukaryote, preferably yeast and algae.

23. The method of any one of claims 1 to 22, wherein the reporter sequence is or comprises a reporter gene which is an antimicrobial resistance gene, and wherein assessing gene expression comprises contacting the host cells with an antibiotic.

24. The method of any one of claims 1 to 22, wherein the reporter sequence is or comprises a reporter gene which encodes a protein that is or generates a chromogenic marker, preferably a fluorescent marker, and wherein assessing gene expression comprises detecting a host cell comprising said chromogenic marker.

25. The method of claim 24, wherein the reporter gene encodes a fluorescent protein, preferably YRP, RFP or GFP, mCherry or luciferase.

26. The method of claim 24 or 25 wherein detection comprises flow cytometry.

27. The method of any one of claims 1 to 26, wherein the library of nucleic acid molecules comprises at least 110.sup.9, 110.sup.12, 110.sup.15 or 110.sup.18 nucleic acid molecules.

28. The method of any one of claims 1 to 27, wherein the library of host cells comprises at least 110.sup.4, 110.sup.6, 110.sup.8, 110.sup.10 or 110.sup.12 host cells, or wherein gene expression is assessed in at least 110.sup.4, 110.sup.6, 110.sup.8, 110.sup.10 or 110.sup.12 host cells.

29. A library of nucleic acid molecules for use in a method as defined in any one of claims 1 to 28 for identifying a factor which modulates gene expression, wherein said library comprises at least 110.sup.6 nucleic acid molecules, wherein each nucleic acid molecule comprises a stochastic sequence of at least 50 nucleotides, optionally wherein each nucleic acid molecule also comprises an identical pre-determined and fixed sequence either adjacent or within the stochastic sequence, wherein said pre-determined and fixed sequence does not include a promoter sequence itself capable of initiating transcription of a gene.

30. The library of nucleic acid molecules of claim 29, wherein the nucleic acid molecules are double-stranded.

31. A library of test constructs, wherein said library comprises at least 110.sup.6 test constructs, each of which comprises a different nucleic acid molecule positioned upstream of a reporter sequence, wherein each nucleic acid molecule comprises a stochastic sequence of at least 50 nucleotides, optionally wherein each nucleic acid molecule also comprises an identical pre-determined and fixed sequence either adjacent or within the stochastic sequence, wherein said pre-determined and fixed sequence does not include a promoter sequence itself capable of initiating transcription of a gene, and wherein each expression vector does not comprise a further separate sequence upstream of the reporter sequence that can modulate expression of the reporter sequence.

32. A library of host cells produced by introducing test constructs from the test construct library into host cells as defined in claim 1 or 2, wherein each host cell from the host cell library comprises a test construct as defined in any one of claims 13 to 17 or 23-25.

Description

[0176] The invention may be better understood from the Figures, in which:

[0177] FIG. 1 shows a nucleic acid molecule comprising a 200 nt stochastic sequence flanked at its 5 and 3 ends by primer binding sites and restriction enzyme recognition (BsaI) sequences, and comprising an E. coli Shine-Dalgarno sequence 8 nt upstream of the position at which translation of the reporter sequence would be initiated once the nucleic acid molecule is introduced into a nucleic acid construct.

[0178] FIG. 2 shows an alignment of the sequences of 20 nucleic acid molecules identified through methods described herein, and shows that stochastic sequences are correctly provided in the test constructs and are able to drive gene expression, and that consensus sequences shared by multiple nucleic acid molecules may be identified.

[0179] FIG. 3 shows the structural overview of synthetic DNA constructs. A, constructs including fixed Shine-Dalgarno sequence (GGAG (SEQ ID NO:45); B, only the random sequence with no fixed sequence.

EXAMPLES

Example 1Beta Lactamase Reporter Sequence

[0180] A library of single-stranded nucleic acid molecules were generated by solid phase synthesis, each comprising a 200 nucleotide stochastic sequence and a 4 nucleotide pre-determined and fixed Shine-Dalgarno sequence for E. coli within the stochastic sequence. Each single-stranded nucleic acid molecule comprised primer binding sites at its 5 and 3 ends to allow PCR amplification, and also included BsaI restriction sites (see FIG. 1) to allow cloning into suitable vectors, e.g. a test construct containing a beta lactamase reporter sequence. The sequence of the nucleic acid molecule including primer binding sites and BSAI restriction sites is provided in SEQ ID NO:1. The reverse complement sequence is provided in SEQ ID NO:2. Following PCR amplification, double-stranded nucleic acid molecules were cloned into nucleic acid constructs to provide a library of test constructs which could be screened to assess the expression of the reporter sequence. The library of test constructs were introduced into E. coli DH5 host cells, and gene expression was assessed by plating cells on plates comprising different concentrations of ampicillin.

[0181] Twenty colonies were randomly picked and were subjected to DNA sequencing using the Bla-Rev primer (SEQ ID NO:30), leading to the promoter-5 UTR sequences listed in FIG. 2, (performed at GATC Biotech, Germany). As expected, the sequencing results indicate the presence of different stochastic sequences in the stretch of DNA preceding the coding sequence for the beta lactamase gene. None of the sequences are identical, indicating that the screen for stochastic sequences was successful. The sequences are set forth in SEQ ID NOs:3-22.

TABLE-US-00001 TABLE 1 Promoter sequence SEQ ID NO: 23892648-6 3 23885241-6 4 23885172-6 5 23883897-6 6 23884068-6 7 23883672-6 8 23883765-6 9 23883741-6 10 23885367-6 11 23883717-6 12 23883861-6 13 23883693-6 14 23885085-6 15 23892801-6 16 23883903-6 17 23887647-6 18 23894490-6 19 23884065-6 20 23892918-6 21 23885328-6 22 Consensus 23, 76

[0182] Multiple alignments of the sequenced stochastic sequences allowed consensus sequences to be identified, which correlated with high levels of expression of the reporter sequence. A portion of a consensus sequence is set forth in SEQ ID NOs:23 and 76 (representing the consensus sequences at the 5 and the 3 ends of the nucleic acid molecule, respectively). A Shine Dalgarno ribosome binding site (GGAGSEQ ID NO:45) is also identifiable in the consensus sequence).

Example 2mCherry Reporter Sequence

[0183] The same library of nucleic acid molecules as was used in Example 1 was prepared, and nucleic acids form the nucleic acid library were cloned into a nucleic acid construct comprising a mCherry reporter sequence and transformed into E. coli cells as above. Transformed cells were screened by plating cells of plates comprising kanamycin. Following overnight incubation, 86 colonies were randomly picked and grown in 96 well plates containing a suitable growth medium. Expression levels of mCherry (red fluorescence protein) in overnight-grown cultures were assessed by fluorometric assay.

[0184] The ability to identify a factor which modulates gene expression in a further bacterial species (Pseudomonas putida KT2440) was also assessed. A library of test constructs was prepared as above and introduced into P. putida KT2440 cells, which were screened for the presence of the test construct by plating the cells on plates comprising kanamycin as above. A broad-host range replicon was used, allowing the same nucleic acid library to be screened in multiple hosts. Expression levels of mCherry in overnight-grown cultures were assessed by the same fluorometric assay, leading to identification of novel expression-mediating StoNuSeqs in this host.

Materials and Methods

Synthesis of Single-Stranded StoNuSeq (Stochastic Nucleotide Sequence)

[0185] The 265 nucleotide long stochastic nucleic acid sequence (StoNuSeq) synthetic DNA was ordered from IDT (Belgium) as a four nmole Ultramer DNA Oligo (i.e. single-stranded).

Generation of Double-Stranded StoNuSeq (ds-StoNuSeq)

[0186] The 265 nt long single-stranded StoNuSeq (ss-StoNuSeq) harbours the BioBrick prefix and suffix on its both ends as adaptors, 5 and 3 ends, respectively (FIG. 1). The ds-StoNuSeq was generated by using the primers BB-Prefix-Fwd (SEQ ID NO:24) and BB-Suffix-Rev (SEQ ID NO:25) in 10 cycles of PCR. The low number of cycles was chosen to prevent the reduction of complexity of the initial ss-StoNuSeq during PCR amplification.

PCR Parameters

[0187] 98 C. for 30 seconds
(98 for 10 seconds, 60 C. for 20 seconds, 72 C. for 20 seconds)10
Construction of Bsa1-Free pUC19

[0188] The only occurring Bsa1 sequence recognition site within the beta-lactamase gene in pUC19 was eliminated by altering the sequence from GGTCTC to GGTCCC without affecting the amino acid sequence. For this the pUC19 vector was amplified using the primer pair Bsa1-Rev (SEQ ID NO:26)/Bsa1-Fwd (SEQ ID NO:27), with the following PCR parameters:

98 C. for 30 seconds
(98 C. for 10 seconds, 62 C. for 20, 72 C. for 80 seconds)25

[0189] The double-stranded PCR product was then transferred to E. coli DH5a using the in vivo homologous recombination method (Bubeck et al. 1993. Nucleic Acids Research 21, 3601-3602), establishing the Bsa1-free pUC19 plasmid.

Ampicillin Screening

[0190] The ds-StoNuSeq harbours two unique Bsa1 restriction enzyme recognition sequences. Upon digestion the ds-StoNuSeq fragment was cloned into the Bsa1-free pUC19, and was transformed to E. coli DH5a. The resulting library was plated out on LA plates with varying concentrations of ampicillin (from 10 to 100 g/mL).

mCherry Screening in E. coli DH5a

[0191] For screening by measuring mCherry fluorescence, three Bsa1 sites in the pHH100-mCherry plasmid had to be eliminated. By using the primer pairs V-Fwd-pHH100 (SEQ ID NO:31)/V-Rev-pHH100 (SEQ ID NO:32) and I-Fwd-pHH100 (SEQ ID NO:33)/I-Rev-pHH100 (SEQ ID NO:34) two PCR products were generated and were transferred to E. coli DH5a by using the in vivo homologous recombination method (Bubeck 1993 ibid). The resulting BsaI-free-pHH100 was amplified using the primer pair Bsa1-mCh-Fwd (SEQ ID NO:35)/Bsa1-mCh-Rev (SEQ ID NO:36). Upon Bsa1 digestion of both insert and vector, the insert was ligated into the vector and transferred to E. coli DH5a. The transformants carrying the constructed vector were plated out on LA-Kan (50 g/mL) plates, creating the StoNuSeq-mCherry library.

mCherry Screening in Pseudomonas putida KT2440

[0192] For screening of expression in P. putida KT2440 cells, the ligation mixture used for mCherry screening in E. coli (described above) was also transferred to P. putida KT2440, by electroporation, similarly to the procedure described for E. coli DH5a (see above). Transformants carrying the constructed vector were selected on LA-Kan (50 g/mL) plates, creating the StoNuSeq-mCherry library in P. putida.

Primers Description:

[0193]

TABLE-US-00002 SEQIDNO:28 Bsa1-Bla-Rev SEQIDNO:29 Bsa1-Bla-Fwd

Example 3

Overview

[0194] Utilising the SUPERAPP technology we have created functional synthetic promoter-5 UTRs, (ProU)s, in seven different microorganisms, across the bacterial and eukaryotic domains of life (Table 2).

Gene Expression Measurement

[0195] For the determination of gene expression two main methods have been used: (i) agar-based assays, or (ii) fluorescent protein measurements.

[0196] The functional determination of gene expression from the reporter genes AMP, APR, CHL, KAN, KANT, TRP were performed on agar-based assays. Libraries of cells were plated on agar medium containing the antibiotics (AMP, APR, CHL, KAN, KANT) or amino-acid (TRP) based on the host-reporter gene. The growth of colonies on these medium indicate the presence of functional ProU sequences. In the case of S. albus and S. lividans cells were also plated on agar plates with higher concentrations of APR (250 and 500 g/mL). Colonies identified on agar plates containing 250 and 500 g/mL APR indicate stronger expression originating from the artificial ProU sequences.

[0197] Fluorescent-based measurements were performed with strains Escherichia coli and Pseudomonas putida expressing RFP. For these measurements colonies with visible red colours were picked from agar plates and were inoculated into 96-well microplates containing LB (with 50 g/mL KAN) and were incubated overnight at 37 C. with 800 rpm agitation. After 18 hours of incubation the RFP expression levels were measured utilising a Tecan fluorescence microplate reader (Tecan).

TABLE-US-00003 TABLE 2 The list of hosts and reporter genes used for the identification of functional ProUs. Selection markers Hosts AMP APR CHL KAN KAN.sup.T RFP YFP TRP B, G () Escherichia coli Pseudomonas putida Thermus () thermophilus B, G (+) Streptomyces () albus Streptomyces () lividans Corynebacterium () glutamicum E Saccharomyces () cerevisiae B, Bacteria; E, Eukaryote; G (), Gram-negative; G (+), Gram-positive; AMP, ampicillin; APR, apramycin; CHL, chloramphenicol; KAN, kanamycin; KAN.sup.T, thermostable kanamycin; RFP, red fluorescent protein; YFP, yellow fluorescent protein; TRP, tryptophan; , confirmed; (), work in progress.

Synthesis of Single-Stranded Random Nucleotide Sequences (RaNuSeq)

[0198] The synthetic DNA used in creating libraries was ordered from IDT (Belgium) as a four nmole Ultramer single-stranded DNA Oligo. Two different versions of the synthetic DNA was ordered: (A) this version has the fixed GGAG (SEQ ID NO:45) sequence in between the two stretches of random DNA, N(200) and N(7); (B) this version has only the random DNA that is of 200 nt, N(200) (FIG. 3).

[0199] The adapters on either end of random DNA provides two functions: Firstly, they are allowing the functional immobilisation of the oligo during the chemical synthesis; secondly, once the single stranded DNA is synthesised these adapters are used to generate the complementary strand by PCR. Adapters also harbour TypeIIS restriction enzyme recognition sequences (BsaI) that are utilised for various downstream applications. The placement of this random sequence directly upstream of the coding sequence has an important implication as it leads to the creation of both promoters and 5 UTR sequences that are functional in combination with the coding sequence. In terms of adapter functionality, adapter 2 can be excised from the double stranded DNA and a gene specific adapter can be ligated depending on the coding sequence of the gene of interest. This allows the utilisation of synthetic DNA libraries for various constructs.

Generation of Double-Stranded RaNuSeq (ds-RaNuSeq)

[0200] The two adapter sequences on either end of DNA include the BioBrick prefix and suffix sequences, respectively (FIG. 3). The ds-RaNuSeq was generated by using the primers BB-Prefix-Fwd and BB-Suffix-Rev in 10 cycles of PCR. The low number of cycles was chosen to prevent the reduction of complexity of the initial ss-RaNuSeq during PCR amplification.

PCR parameters: (9830)+[(9810),(6020),(7220)]10

TABLE-US-00004 TABLE 3 The synthetic DNA and the cloning method utilised for the construction of DNA libraries. Hosts Synthetic DNA design Cloning method Escherichia coli A for RFP/B for AMP, Gibson for RFP/ APR, CHL, KAN, Bsal for the rest KAN.sup.T Pseudomonas putida A for RFP Gibson Thermus thermophilus A for KAN.sup.T Gibson Streptomyces albus B for APR Bsal Streptomyces lividans B for APR Bsal Corynebacterium B for CHL Bsal glutamicum Saccharomyces cerevisiae B for TRP Bsal AMP, ampicillin; APR, apramycin; CHL, chloramphenicol; KAN, kanamycin; KAN.sup.T, thermostable kanamycin; RFP, red fluorescent protein; TRP, tryptophan.
Escherichia coli

[0201] In the construction of all libraries E. coli was used a cloning host. For the specific selection of dual-host functionality it was also used as an expression host except for the yeast specific TRP (Table 2).

[0202] E. coli cells were grown in Lysogeny broth (per L: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar for solid plates) at 37 C.

DNA ManipulationsPlasmid/Vector Constructions

[0203] Two general approaches were used to create the libraries: BsaI- or Gibson assembly-based.

BsaI Restriction Based Cloning

[0204] For this restriction and ligation based cloning strategy, the plasmid was amplified with a primer pair that excludes the promoter upstream of the gene of interest. At the same time, the primers introduce a type II restriction site which is recognized by BsaI. The restriction site overhangs are designed in a way that the restriction site itself will be cleaved off, leaving an NATG overhang at the start of the gene of interest and an overhang. Vector and insert was ligated using either T4 ligase or quick ligase and a vector to insert ration of 1:7.

Gibson Assembly

[0205] Gibson assembly mix was made according to Gibson et al., 2009(DOI: 10.1038/nmeth.1318)

[0206] Use equimolar amounts (or 1:2 ratio) of both purified vector and insert was used (100 ng) and a volume of 5 L, added to a volume of 15 L of Gibson assembly mix.

[0207] In this procedure, the primer pairs exclude the natural promoter in front upstream of the gene of interest. The reverse primer also introduces the BB Prefix, upstream of the promoter region to be used as an homologue sequence Gibson assembly cloning.

[0208] For the downstream part, the library has to be fitted with an adapter that is specifically designed for the gene of interest.

Adapter Attachment Protocol

[0209] Upon BsaI digestion the DNA library carries a NATG overhang (SEQ ID NO:75). Complementary oligos can be designed with suitable overhang which can be ligated. This adapter attachment allows Gibson assembly. Overhangs can be between 15 and 30 bp long.

Phosphorylation of the Adapters

[0210] 7 L oligo 1 (700 pmol) use 100 M primer dilution (100 M (=100 pmol/L))
7 L oligo 2 (700 pmol) use 100 M primer dilution (100 M (=100 pmol/L))

1.5 L T4 Ligase Buffer

0.8 L PNK

Activation for 30 min at 37 C.

[0211] 30 min at 65 C. for heat inactivation
Add 1 L of 4 M NaCl and run the annealing program Anneal on the PCR2 machine.
Make dilutions up to 1:1000. Total amount is 1400 pmol/17.3 L=80.9 pmol/L undiluted
95 C. 10 m, 80 C. 2 m, 75 C. 2 m, 70 C. 3 m, 65 C. 5 m, 60 C. 10 m, 55 C. 10 m. 50 C. 5 m, 45 C. 3 m, 40 C. 2 m, 35 C. 1 m 30 C. 1 m, 25 C. 1 m, 20 C. 1 m, 4 C. hold.

Ligation of the Gene Specific Adapter

5 L Quick Ligase Buffer

[0212] 30 ng (200 fmol) vector library del BsaI
1 L of 1:100 dilution (800 fmol) insert adapter (1:100 dilution, gives a 1:4 molar ration vector to insert, results in 25 mM NaClcommercial ligases can handle up to 70 mM NaCl)

Quick Ligase 0.5 L

H.SUB.2.O Fill to 10 L

[0213] Incubate at RT for 5 min and then use desired amount for PCR (have used up to 5 L)
PCR may be done with either Q5 or Taq, Taq adds some As, but these are removed in the assembly process:

10 L Q5 Buffer

[0214] 1 L dNTPs
2.5 L library_biobrick_del_F (SEQ ID NO:71)/AII_biobrick_F(SEQ ID NO:70)
2.5 L rv adapter

0.5 L Q5 PoI

5 L Template (QL)

28.5 L H.SUB.2.O

Run PCR (Iti):

98 C. 30 s

[0215] 98 C. 10 s (do 10 cycles)
20 C. s (adjust this temperature to your primer pair)
72 C. 20 s (cycle ends)

72 C. 1 min

[0216] 4 C. hold

[0217] Run on gel to verify the size (250 nt with 200N del BsaI library, depending on adapter size)

Heat Shock Transformation

[0218] Take chemically competent DH5 E. coli, 100 L aliquots from the 80 C. freezer (10.sup.6 cfu). Let chill on ice for 10 minutes. Add an amount 1-200 ng for a plasmid or about 40-50 ng DNA from a Gibson mix (half of the mix which in total should not be more than 100 ng).

Incubate on ice for 30 min

[0219] Heat shock at 42 C. for 45 seconds
Chill on ice for 2-5 minutes
Add 900 L LB media
Shake at 27 C. for one hour. Take 200 L per medium agar plate or whole transformation batch (1 mL) for a big agar plate and spread out.
Incubate over night at 37 C.
Pseudomonas putida KT2440

[0220] P. putida cells were grown in Lysogeny Broth (or agar 15 g/L) at 30 C.

Plasmid/Vector Detail

[0221] Plasmid DNA: pHH100 vector, which is suitable for gene expression in organisms E. coli and P. putida. Kanamycin resistance which is expressed in both host organisms and is used as a selective pressure. The plasmid also contains a gene that codes for an mCherry protein that can be expressed by both organisms.

DNA ManipulationsPlasmid/Vector Construction

[0222] For this part of the work, Gibson assembly was used (see E. coli section for general procedure). The PCR uses the primer pair: Pp_pHH100_mCherry_F (SEQ ID NO:67) and Pp_pHH100_mCherry_R (SEQ ID NO:68) which creates a backbone that excludes the natural mCherry promoter and introduces the BioBrick Prefix sequence. Note that for a successful PCR, the linearized plasmid had to be used. The plasmid was digested using the NdeI site which is just upstream of the mCherry gene. The backbone was DpnI treated and purified and used in the Gibson assembly together with the suitable library. The Gibson mix is transferred into the cloning host E. coli via heat shock transformation and grown on LB plates containing kanamycin. The library size is about 3000 colonies per 10 L Gibson mix. From this library, about 200 clones were picked that were appearing red to naked eye. The plasmids were isolated from the whole library and transferred into P. putida by electroporation.

DNA Transformation Details

[0223] An overnight culture of the recipient strain was diluted in 1:100 in a rich medium with appropriate antibiotics. One mL of culture was used for every transformation. Cultures were grow at 30 C. with shaking for 2-4 h, until an OD600 0.3. For each transformation, 1 mL of culture was pelleted in a microcentrifuge tube. Pellets were resuspended in 1 mL cold sterile water or 10% glycerol, and centrifuged once more and resuspended in 1 mL of water or 10% glycerol. Centrifuged once more and pellet was resuspended in 50 L of cold sterile water or 10% glycerol.

To Transform Electrocompetent Cells

[0224] Mix an aliquot (50-70 L) of cells with 1-2 L DNA in a low ionic strength buffer/water (dialyze or EtOH-precipitate ligations etc.)
Place in pre-chilled cuvette
Electroporate (2.5 kV for 2 mm gap or 1.8 V for 1 mm gap; 200, 25 F), Time constant should be 4 msec
Immediately add 1 mL of SOC or LB to cell/DNA mix and place in test tube
Grow at 30 C. for 1.5 hours
Plate onto selective media
Grow overnight at 30 C.

Sequencing

[0225] For sequencing, plasmids were isolated with the Plasmid Miniprep kit from Qiagen and were sequenced using the primer Pp_pHH100_mCherry_Seq.

Thermus thermophilus

[0226] A knockout strain of Thermus thermophilus HB27 with high transformation efficiency (Genome accession number: AE017221), HB27ago, was provided by Dr. Jos Berenguer (Universidad Autnoma de Madrid) and used in this study.

Growth Conditions

[0227] Thermus Broth (TB) was used for T. thermophilus strain propagation and assays, which contains the following: bactotryptone (8 g/L), yeast extract (4 g/L) and NaCl (3 g/L), all dissolved in mineral water. 1.5% agar was added to the TB medium for growth on plates. A gradient range of Kanamycin (30 g/ml, 60 g/ml, 90 g/ml) were added to the growth medium when selection is required.

[0228] Stock pellet of T. thermophilus from 20 C. was thawed at room temperature, before the addition of 1 mL of TB and in subsequent transferred to a 100-150 mL flask with 20 mL of TB. The strain was cultivated in a 65 C. shaker under mild shaking (150 rpm). To avoid plates from drying, TB plates were incubated in a moisturized Tupperware box in a 65 C. incubator.

Plasmid/Vector Details

[0229] The E. coli/T thermophilus shuttle vector, MK184 (Jos Berenguer, 2007, DOI: 10.1111/j.1365-2958.2007.05687.x), provides the thermostable Kanamycin resistance as the screening reporter.

DNA Manipulations and Plasmid/Vector Construction

[0230] The pSLPa promoter of the thermostable kanamycin marker was excluded via PCR using the primers pair Tt_pMK184_kan_F (SEQ ID NO:61) and Tt_pMK184_kan_R (SEQ ID NO:62). The PCR was carried out with CloneAMP HiFi PCR premix (Takara Bio, Inc.) and reagents concentrations, Tm were set (55 C.) according to the manufacturer instructions.

[0231] The site of the original promoter is replaced with a randomized sequence library via Gibson assembly. A BioBrick Prefix homology site is introduced via the overhang of the reverse primer on the vector, which assemble to one end of the randomized sequence library. On the other end, the adaptor-attached randomized sequence library contains around 20 nucleotide homologies to the reporter gene. Gibson assembly of the promoter-less vector and adaptor-attached randomized sequence library was carried out using a home-made Gibson reaction mix (Gibson et al., 2009, Nature, DOI: 10.1038/nmeth.1318). The vector/Insert ratio was adjusted by sequence length to 1:1 and up to a 100 ng of DNA was transformed to E. coli strain DH5, which serves as the cloning host and used for the first round of synthetic promoters screening. A Shine-Dalgarno sequences containing library was used in this screen.

DNA Transformation Details

[0232] Overnight T. thermophilus strain culture was re-inoculated in fresh pre-warmed TB medium at a dilution of one over fifty. The strain was cultivated in a 65 C. shaker until an OD.sub.550 nm of 0.4 was reached. 0.8 mL of the culture was aliquoted to a new 12 mL tube, follow by an addition of 200 ng of plasmids. The mixture was incubated further at 65 C. with shaking for 4 hours, after which it was plated on selective TB plates. Colonies were isolated after an overnight incubation at 65 C.

Phenotypic Screening and Promoter Activity Confirmations

[0233] To isolate cross species synthetic promoters between E. coli and T. thermophilus, 10 out of 20 L of the Gibson assembly mix was first chemically transformed to E. coli DH5 under a Kanamycin selection of 50 g/mL. Around 1,500 colonies were obtained with 3 to 4 rounds of transformations. The cell lawn was then scraped and the plasmids were isolated with QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturers and later quantified with Nanodrop.

[0234] 200 ng of the plasmids mix with functional promoters from E. coli were transformed to T. thermophilus. The transformants were challenged by a series of kanamycin concentrations (30 g/ml, 60 g/ml, 90 g/ml) and the resulted colonies were picked and further purified with streaking.

Sequencing

[0235] Purified colonies were re-streaked on TB agar with the basal kanamycin selection (30 g/mL). After an overnight incubation, the cell lawn was scraped and the plasmid was isolated with QIAprep Spin Miniprep Kit (Qiagen). The plasmid and a reverse sequencing primer, Tt_pMK184_seq (SEQ ID NO:63), was together sent for LIGHTRUN sequencing (GATC Biotech). The obtained sequences were checked and analysed using Genome Compiler software.

Streptomyces

Bacterial Strains and Growth Conditions

[0236] The bacterial strains used in this study are listed in Table 4.

TABLE-US-00005 TABLE 4 Bacterial strains and plasmids used in this study. Bacterial strains and plasmids Description Reference S. albus J1074 Derivative of S. albus DSMZ Chater and Wilde, 1980 40313, isoleucine and valine DOI: 10.1099/00221287- auxotrophic, deficient of 116-2-323 Sa/GI-based restriction modification system. Widely used for heterologous protein/antibiotic production. S. lividans TK24 Plasmid-free derivative Hopwood et al., 1982 strain of S. lividans 66. doi: 10.1099/00221287-129- Model Streptomyces strain 7-2257 routinely used for heterologous protein production. E. coli DH5 Standard cloning. E. coli S17.1 Conjugative transfer of DNA to Streptomyces spp. E. coli C2984 High efficiency turbo NEB competent cells for library construction. pKC1218 Conjugative E. coli-Streptomyces Bierman et al., 1982 shuttle vector. https://doi.org/10.1016/0378- SCP2* Streptomyces 1119(92)90627-2 replicon, pMB1 E. coli replicon, aac(3)IV gene conferring apramycin resistance. pKC1218_kan pKC1218-derivative with This study. inserted aph(3) cassette between oriT and SCP2* conferring kanamycin resistance. pKC1218_kan-200N pKC1218_kan with 200N This study. library inserted directly upstream of aac(3)IV start codon. pKC-P pKC1218_kan with This study. promoterless aac(3)IV gene.

[0237] E. coli strains were grown in Lysogeny broth. When required, antibiotics were added to cultures at the following concentrations: 50 g/mL kanamycin, 50 g/mL apramycin. For sporulation S. lividans TK24 was grown at 30 C. on ISP4 agar (BD Difco ISP medium 4), S. albus J1074 at 30 C. on soy flour mannitol agar (20 g/L soy flour, 20 g/L mannitol, 20 g/L agar). Conjugation reactions were performed using the same media supplemented with 10 mM MgCl.sub.2 at 30 C.

Recombinant DNA Techniques

[0238] Plasmid DNA from E. coli was isolated using standard protocols. Restriction enzymes and molecular biology reagents were used according to the manufacturers' instructions (NEB, England).

TABLE-US-00006 TABLE 5 Primers used in this study. Sequence Primers 5-3 Purpose pKC_fwd_kan SEQ ID Cloning the aph(3) gene into pKC1218. NO: 37 pKC_rev_kan SEQ ID Cloning the aph(3) gene into pKC1218. NO: 38 Bsal_Apr5f_new SEQ ID Introduction of Bsal restriction sites for NO: 39 cloning of 200N library upstream of aac(3)IV start codon. Bsal_oriV3r SEQ ID Introduction of Bsal restriction sites for NO: 40 cloning of 200N library upstream of aac(3)IV start codon. BB_Prefix_Fwd SEQ ID Amplification of 200N library (without NO: 24 SD) for cloning upstream of promoter-less aac(3)IV gene. BB_Suffix_Rev SEQ ID Amplification of 200N library (without NO: 25 SD) for cloning upstream of promoter-less aac(3)IV gene. 5511-F SEQ ID Colony PCR NO: 41 234_R SEQ ID Colony PCR NO: 42 5654_F SEQ ID Sequencing primer NO: 43 apr_5c SEQ ID Sequencing primer NO: 44
Construction of DNA in E. coli

[0239] For selection of functional synthetic promoters/UTRs in E. coli and Streptomyces, a shuttle expression plasmid based on pKC1218 (Table 1), a conjugative vector with low copy Streptomyces replicon (SCP2* rep), pMB1 E. coli replicon and apramycin resistance cassette (aac(3)IV) was modified as follows.

[0240] The pKC1218 backbone was amplified using primers pKC_rev_kan and pKC_fwd_kan (Table 5) introducing 40 bp homology overhangs to a 1100 bp SalI-fragment of pTA16 containing the aph(3) kanamycin resistance gene, and the cassette was cloned between oriT and SCP2 replicon by in vivo homologous recombination in E. coli DH5 (Bubeck et al. 1993, Nucleic Acids Research, 21, 3601-3602, yielding pKC1218_kan.

[0241] pKC1218_kan was linearized by PCR using primers BsaI_Apr5f_new and BsaI_oriV3r (Table 5) introducing BsaI-overhangs on both ends. The product was phosphorylated using T4 PNK and re-ligated and the circularized product digested with BsaI. A library of 200 nucleotide long random sequences was amplified by PCR using primers BB_Prefix_Fwd and BB_Suffix_Rev (Table 2), digested with BsaI and ligated directly upstream of the promoterless aac(3)IV gene, creating a library of 200 nucleotide long randomized promoter plus 5 UTR sequences in pKC1218_kan. This library was then transferred to turbo competent E. coli C2984 cells (NEB) by chemical transformation. Transformants were selected on 14 cm agar plates containing LA supplemented with 50 g/mL kanamycin. The entire population of obtained transformants (lawn on plate) was pooled and plasmid DNA isolated using QuiaPrep Spin Miniprep Kit (Qiagen) according to the manufacturers' instructions. 1 g of plasmid DNA was used to transform chemically competent E. coli S17.1 cells for conjugal transfer of the library to Streptomyces. S17.1 transformants were selected on LA medium supplemented with 50 g/mL kanamycin. To select for promoters/UTRs functional both in E. coli and Streptomyces, transformants were also selected on LA supplemented with 50 g/m apramycin (Lib Am).

Selection of Functional Promoters/UTRs in S. albus J1074 and S. lividans TK24

[0242] For selection of functional synthetic promoters/UTRs based on antibiotic resistance (apramycin) phenotype, the 200N-library was transferred from E. coli S17.1 to S. albus J1074 and S. lividans TK24 by intergenic conjugation. Conjugation reactions were performed as described previously (Kieser et al., 2000) with minor modifications. S17.1 library transformants (ca. 20.000 transformants for LibAm and Lib Km, respectively) were pooled by adding 3 mL LB medium to a 14 cm agar plate and the cells brought into suspension using a sterile glass rod. 100 L of the obtained suspensions was used to inoculate 25 mL LB medium and incubated at 37 C. for ca. 2.5 h until the cells had reached an OD.sub.600 of 0.4. The cells were centrifuged at 2.000g for 5 min at room temperature, and the obtained pellet re-suspend in 2 mL fresh LB medium and placed on ice. Spore suspensions of two freshly sporulated plates of S. albus and S. lividans were prepared using 4 mL sdH.sub.2O and the obtained suspensions filtered through sterile cotton wool. 50 l of these spore suspensions were added to 500 l 2YT medium (16 g/L Tryptone, 10 g/L, Yeast Extract, 5 g/L NaCl) and incubated at 50 C. for 5 min to induce germination. The spore suspensions were cooled under running water before 500 l of E. coli suspension was added, and the resulting suspension was mixed by inversion before spreading it onto two 9 cm agar plates (SFM+10 mM MgCl.sub.2 for S. albus, and ISP4+10 mM MgCl.sub.2 for S. lividans). Conjugation plates were incubated at 30 C. for 14-16 h before overlaying them with antibiotic solutions yielding final concentrations of 30 g/mL nalidixic acid and 50/250/500 g/mL apramycin in agar media. The plates were then further incubated at 30 C. until exconjugants appeared (2-3 d). Single exconjugant colonies were transferred to fresh plates supplemented with corresponding concentrations of apramycin for further analysis by colony PCR and sequencing of the 200N region.

Sequence Analysis of Functional Synthetic Apramycin Promoters/UTRs

[0243] Single colonies of exconjugants were subjected to colony PCR using primers 5511_F and 234_R (Table 2) amplifying a 710 bp fragment surrounding the 200N region. Single colonies were picked into 100 mL of 200 mM lithium acetate and 1% SDS and incubated at 70 C. for 5 min. 300 mL of 96% ethanol were added, the suspension vortexed and centrifuged at 15.000g for 3 minutes. The pellet was washed with 70% ethanol, dried and dissolved in 20 mL sterile deionized water. 1 mL of the resulting solution was used as template for PCR reactions using Taq polymerase (NEB). Amplicons of the expected size were extracted from 0.8% agarose gels and purified using the QiaQuick gel extraction kit (Qiagen) according to the manufacturers' instructions, and send for sequencing using sequencing primers 5654_F or apr_5c (Table 2). [0244] Results
Screening of 200N Library for Functional Promoters/UTRs in S. albus J1074

TABLE-US-00007 TABLE 6 Number of exconjugants obtained per plate after transferring the 200N library (pKC1218_kan-200N) to S. albus J1074 and screening for functional promoters/UTRs using different concentrations of apramycin (cfu/plate). Overlay Overlay Overlay Overlay Library Dilution with Km with Am with Am with Am type plated 50 50 500 2000 Lib Km 0 420 220 2 0 Lib Km 10.sup.1 31 91 1 0 Lib Km 10.sup.2 1 3 0 0 Lib Km 10.sup.3 0 0 0 0 Lib Am 0 800 640 6 0 Lib Am 10.sup.1 30 180 7 0 Lib Am 10.sup.2 2 8 0 0 Lib Am 10.sup.3 0 0 0 0 Lib Km = transfer of 200N library from C2984 to S17.1, selection of transformants with Km50; Lib Am = transfer of 200N library from C2984 to S17.1, selection of transformants with Am50.

[0245] 200N exconjugants for S. albus J1074 were successfully obtained upon selection with up to 500 g/mL apramycin, while cells carrying pKC1218_kan with the wild-type apramycin promoter were only able to grow on medium supplemented with up to 50 g/mL apramycin. The apramycin promoterless version of pKC1218_kan served as a negative control and S. albus exconjugants carrying this plasmid were not obtained when selecting with 50 g/mL apramycin.

Screening of 200N Library for Functional Promoters/UTRs in S. lividans TK24

[0246] 200N exconjugants for S. lividans were successfully obtained upon selection with up to 500 g/mL apramycin. Exconjugants carrying pKC1218_kan with the wild-type apramycin promoter were also able to grow on medium supplemented with up to 500 g/mL apramycin. The apramycin promoterless version of pKC1218_kan supported growth of S. lividans exconjugants when selected with up to 50 g/mL but not at any higher concentration tested (250 g/mL and 500 g/ml).

TABLE-US-00008 TABLE 7 Number of exconjugants obtained per plate after transferring the 200N library (pKC1218_kan-200N) to S. lividans TK24 and screening for functional promoters/UTRs using different concentrations of apramycin (cfu/plate). Overlay Overlay Overlay Overlay Library Dilution with Km with Am with Am with Am type plated 50 50 500 2000 Lib Km 0 19 80 30 Lib Km 10.sup.1 135 61 23 Lib Am 0 0 50 255 Lib Am 10.sup.1 325 200 122 Lib Km = transfer of 200N library from C2984 to S17.1, selection of transformants with Km50; Lib Am = transfer of 200N library from C2984 to S17.1, selection of transformants with Am50.

TABLE-US-00009 TABLE 8 Transfer of pKC1218_kan (with native apramycin promoter) and pKC-P (=PCR-amplified, phosphorylated and religated pKC1218_kan without AM promoter) from S17.1 to S. albus and S. lividans, (cfu/plate). Km50 Am50 Am250 Am500 S. albus/pKC1218_kan, 6400 7000 0 0 10.sup.0 S. albus/pKC1218_kan, 105 300 0 0 10.sup..sup.1 S. albus/pKC-P, >>6400 0 0 0 10.sup.0 S. albus/pKC-P, 300 0 0 0 10.sup.1 S. lividans/pKC1218_kan, 240 200 160 100 10.sup.0 S. lividans/pKC1218_kan, 7 3 2 0 10.sup.1 S. lividans/pKC-P, 10.sup.0 2500 2500 0 0 S. lividans/pKC-P, 10.sup.1 450 25 0 0
Corynebacterium glutamicum

[0247] C. glutamicum wild type ATCC 13032 cells were grown in Brain heart infusion medium (Brain heart infusion mix: 37 g/L, 91 g/L of sorbitol [in broth medium only] at 30 C.

DNA ManipulationsPlasmid/Vector Construction

[0248] BsaI Restriction Based Cloning in Vector pXMJ19.

[0249] pXMJ19 is a shuttle vector that is suitable for replication in E. coli and C. glutamicum. The chloramphenicol resistance gene works in both organisms. Amplification of the whole plasmid under exclusion of CHL promoter with the primer pair: Cg_pXMJ19_Chl_F and Cg_pXMJ19_Chl_R. This same primer pair introduces BsaI restriction sites as overhangs and creates a backbone of 4300 nt. After the PCR, the product is digested with DpnI and BsaI in CutSmart at 37 degree for 3 hrs and cleaned up. The library is being amplified (without SD) and cut with BsaI and purified. For the T4 ligation over night at 16 degree use: 60 ng of backbone and 20 ng of insert (1:7 backbone to insert ratio). Heat inactivate the next day and use 10 L for transformation into E. coli gives 1000 transformants/10 L. library size total 2000.

DNA Transformation Details

[0250] For cloning in E. coli was used following the stranded heat shock protocol for chemical competent cells. For DNA transformation to C. glutamicum, follow the protocol was used:

Inoculate 5 mL BHIS (Brain Heart infusion) and incubate at 30 C. O/N
Inoculate 25 mL BHIS with 500 l pre-culture
Incubate at 30 C. until OD600=1.5
Centrifuge at 4500 rpm for 3-5 min at 4 C. Discard supernatant
Wash cells 2 in 25 mL cold TG buffer (10% glycerol, 1 mM Tris)
Centrifuge at 4500 rpm for 3-5 min at 4 C. Discard supernatant
Wash cells 2 in 25 mL cold 10% glycerol
Centrifuge at 4500 rpm for 3-5 min at 4 C. Discard supernatant
Re-suspend cell in back-flow (400 l) and keep on ice
Add 100 l of cells and up to 1 g DNA to cold electroporation cuvette

Electroporate at 2.5 kV, 25 F

[0251] Add cells to 4 mL BHIS (preheated at 46 C.)
Incubate cells at 46 C. for 6 min
Incubate cells at 37 C. for 1 hr
Incubate cells at 30 C. for 30 min
Centrifuge at 4500 rpm for 3-5 min and plate on selection media
Incubate at 30 C. for 2 days

Phenotypic Screening and Promoter Activity Confirmations

[0252] Screening for expression of CHL resistance gene on CHL 15 plates. Library size in E. coli was 2000, going down to 200 in Cg.

Sequencing

[0253] Plasmids were isolated using the plasmid miniprep kit from QIAGEN. Since C. glutamicum is a gram positive organism, an extra step is required: Make a solution of P1 buffer+15 mg/mL final concentration of lysozyme. Use 350 L of that to incubate with the colony for 3 hours at 37 degree. Plasmid concentrations usually too low to send for sequencing->retransformation into E. coli via heat shock, then isolation from there. For sequencing use the primer: Cg_pXMJ19_Chl_Seq (SEQ ID NO:64).

Saccharomyces cerevisiae

Growth Conditions;

Growth Medium:

[0254] YPD: yeast extract peptone dextrose ready-to-use from Sigma-Aldrich. Use: 50 g in 1 L of distilled water. Autoclave for 15 minutes at 121 C. For agar plates, add 15 g/l.
Contains (g/L):
Bacteriological peptone, 20
Yeast extract, 10

Glucose, 20

[0255] For yeast cultivation prior to transfection use 2YPD

Drop Out Media:

[0256] Make a 10 concentrated stock solution by stirring to suspend 6.8 g yeast nitrogen base powder (Sigma), 5 g glucose, and 5-10 mg of appropriate amino acids in 100 mL water. Warm if necessary to aid solubilization. Filter sterilize and store at 2-8 C. Appropriate amino acids: Add 1.92 g/L of Yeast Synthetic Drop-Out Media Supplements without tryptophan (Sigma)

[0257] Dilute the 10 concentrated stock to a 1 working solution by adding 100 mL concentrated stock to 900 mL sterile water. For plates: Autoclave water plus 15 g/L agar together, add from 10 sterile stock appropriate amount afterwards.

Growth temperature: 30 C.

DNA ManipulationsPlasmid/Vector Construction(s);

[0258] Cloning in E. coli. The vector used was pENZ004 provided by Sara Castao Cerezo from the Institut National des Sciences Appliques de Toulouse|INSA Toulouse, Biosystems and Process Engineering Laboratory (LISBP). pENZ004 is a shuttle vector suitable for cloning in E. coli, which also contains homologous regions for integration into the Site 2 chromosome locus in S. cerevesia. For Gibson cloning, the vector was amplified using primer pair Sc_pENZ004_G_trp_F (SEQ ID NO:52) and Sc_pENZ004_G_trp_R (SEQ ID NO:53). The adapter for the library was created using primer pair Adapter_pENZ004_trp_UpS (SEQ ID NO:73) and Adapter_pENZ004_trp_LoS (SEQ ID NO:74). The adapter library then was amplified using AII_biobrick_F (SEQ ID NO:70) and Adapter_pENZ004_trp_LoS For BsaI cloning, the vector was amplified using primer pair Sc_pENZ004_B_trp_F (SEQ ID NO:57) and Sc_pENZ004_B_trp_R (SEQ ID NO:58).

[0259] There is no direct screening of the library in E. coli, as they are being selected for the plasmid on the plasmid ampicillin resistance.

DNA Transformation Details;

[0260] Homologous recombination in yeast (inspired by Belden et al, 2015, Journal of Microbiological Methods https://doi.org/10.1016/j.mimet.2013.11.013 and Gietz, .sub.2014, Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-0799-1_4)
Inoculate the yeast strain into 5 mL of liquid medium (2YPD) and incubate overnight on a rotary shaker at 200 rpm and 30 C.
Cut desired plasmid to linearize. Use 100 ng and use dirty digestion mix for transformation. Here, cut with Sfil in CutSmart at 50 degree.
Determine the titer of the yeast culture: Pipet 100 L of cells into 900 L of 2YPD in a spectrophotometer cuvet, mix thoroughly by inversion, and measure the OD at 600 nm. An OD600 of 1 equals 310.sup.7 cells/mL.
Add cells to 25 mL of pre-warmed 2YPD to a titer of 510.sup.6 cells/mL, grow for about 4 hrs at 30 degree, shaking until titer reaches 210.sup.7 cells/mL.
Harvest the cells by centrifugation at 3000 g for 5 min, wash the pellet twice in 25 mL of sterile water, and resuspend the cells in 1.0 mL of 0.1 M LiOAc.
Transfer the cell suspension to a 1.5-mL microcentrifuge tube, centrifuge for 30 s, and discard the supernatant.
Resuspend the cells in 100 L per 110.sup.8 cells total of 0.1 M LiOAc and pipet samples of 50 L (about 510.sup.7 cells into aliquots. Keep aliquots on bench top until use or in fridge for up to one week.
Prepare the T Mix (can be done beforehand and kept on ice/water until usage):
Dissolve 2 mg of salmon sperm DNA (Sigma) in 1 mL of TE: 10 mM Tris-HCl, 1 mM Na2 EDTA pH 8.0, using a stir plate overnight at 4 C.
Denature an appropriate sample size of carrier DNA in a boiling water bath for 5 min and chill immediately in an ice/water bath.
Add together:
240 L PEG 3500 (50% [w/v])

36 L LiAc 1.0 M

[0261] 50 L SS carrier DNA (2.0 mg/mL), denaturated
34 L Digested plasmid DNA (100 ng)
360 L Total volume (excluding cells)
Note: Vortex the carrier DNA before pipetting it
Centrifuge yeast cells at top speed for 30 s, remove supernatant.
Add 360 L of T Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
Place the tubes in a shaker at 30 degree for 30 min.
Place the tubes in a floating rack and incubate them in a water bath at 42 C. for 30 min. Invert every 5 minutes.
Microcentrifuge the tubes at top speed for 30 s and remove the T Mix carefully and completely with a micropipettor.
Pipet 400 L of sterile water into the transformation tube. Dissolve the pellet with a sterile micropipette tip to resuspend the cells.
Incubate the plates at 30 C. for 3-4 d and count the number of transformants.

Phenotypic Screening and Promoter Activity Confirmations;

[0262] Phenotype screening for growth on drop out media. Library size in cloning host was in 10.sup.4 range, in yeast then to 10{circumflex over ()}3.
Details on sequencing;
Crude yeast genomic DNA extraction was performed with the following procedure (Kristjuhan et al (2011, Biotechniques, doi: 10.2144/000113672)):

Reagents

[0263] 1. 0.2 M Lithium acetate 1% SDS solution.

2. Ethanol 96-100% and 70%.

Procedure

[0264] 1. Pick one yeast colony from the plate or spin down 100-200 l of liquid yeast culture (OD600=0.4). Suspend cells in 100 l of 200 mM LiOAc, 1% SDS solution.
2. Incubate for 5 minutes at 70 C.
3. Add 300 l of 96-100% ethanol, vortex.
4. Spin down DNA and cell debris at 15 000 g for 3 minutes.
5. Wash pellet with 70% ethanol
6. Dissolve pellet in 100 l of H2O or TE and spin down cell debris for 15 seconds at 15 000 g.
7. Use 1 l of supernatant for PCR.

[0265] The PCR creates a 2000 nt piece from the upstream homologous region to the downstream homologous region using primer pair: Sc_pENZ004_Hom_F (SEQ ID NO:54) and Sc_pENZ004_Hom_R (SEQ ID NO:55). This piece was purified and then sent for sequence analysis with an internal primer: Sc_pENZ004_Seq (SEQ ID NO:56).

[0266] A number of promoters/5 UTR sequences were identified for C. glutamicum (10 sequences), E. coli (146 sequences), P. putida KT2440 (11 sequences), S. cerevisiae (10 sequences), S. albus (16 sequences), S lividans (8 sequences) and T. thermophilus (11 sequences) as described above. A selection of the sequences identified for each organism is provided in Tables 9-15 below.

TABLE-US-00010 TABLE 9 C. glutamicum sequences Clone SEQ ID NO 22EF79 77 22EF75 78 22EF83 79 22EF80 80 22EF97 81

[0267] C. glutamicum sequences identified using a Chloramphenicol reporter gene.

TABLE-US-00011 TABLE 10 E. coli sequences Clone Fluorescence measurement (au) SEQ ID NO E6 2246 82 F3 8034 83 H5 12237 84 J4 30254 85 O3 59961 86

[0268] E. coli sequences identified using an mCherry reporter gene. Fluorescence measurements for each clone are shown as an indication of the expression level seen for each clone

TABLE-US-00012 TABLE 11 P. putida KT 2440 sequences Clone SEQ ID NO: 22EF90 87 22EF86 88 22EF88 89 22EF87 90 22EF85 91

[0269] P. putida KT2440 sequences identified using an mCherry reporter gene.

TABLE-US-00013 TABLE 12 S. cerevisiae sequences Clone SEQ ID NO 22EF21 92 22EF23 93 22EF25 94 22EF27 95 22EF29 96

[0270] S. cerevisiae sequences identified using a TRP1 reporter gene.

TABLE-US-00014 TABLE 13 S. albus sequences Clone Apramycin concentration used for selection SEQ ID NO: 1 50 g/ml 97 2 50 g/ml 98 3 50 g/ml 99 4 50 g/ml 100 2 500 g/ml 101

[0271] S. albus sequences identified using an aac(3)IV reporter gene. Concentrations of apramycin used in the selection of clones are shown.

TABLE-US-00015 TABLE 14 S. lividans sequences Clone Apramycin concentration used for selection SEQ ID NO: 1 500 g/ml 102 2 500 g/ml 103 3 500 g/ml 104 4 500 g/ml 105 5 500 g/ml 106

[0272] S. lividans sequences identified using an aac(3)IV reporter gene. Concentrations of apramycin used in the selection of clones are shown.

TABLE-US-00016 TABLE 15 T. thermophilus sequences Clone SEQ ID NO: K60_C1 107 K90_C4 108 K60_C5 109 K90_C5 110 K60_C20 111

[0273] T. thermophilus sequences identified using a thermostable kanamycin resistance reporter gene.