Engineered cssdna production hosts and phagemids

Abstract

Described herein are engineered bacterial production strains that include engineered phagemids for the efficient production of high quality circular, single-stranded DNA (cssDNA). Also, described herein are DNA molecules that can be used to efficiently produce high quality cssDNA. Accordingly, methods of producing high quality cssDNA are also described.

Description

Abstract This paper describes engineered bacterial production strains containing engineered phage particles for the efficient production of high-quality circular single-stranded DNA (cssDNA). Furthermore, this paper describes DNA molecules capable of efficiently producing high-quality cssDNA. Therefore, methods for producing high-quality cssDNA are also described.

Claims

WHAT IS CLAIMED IS:

1. A production strain comprising a phagemid and at least two phage protein coding sequences integrated into the production strain genome, wherein the production strain can produce cssDNA upon introduction of the phagemid.

2. A circular single-stranded DNA (cssDNA) production system, comprising: at least two phage protein coding sequences integrated into the production strain genome; and a phagemid, wherein the production strain can produce cssDNA upon introduction of the phagemid into the production strain.

3. A circular single-stranded DNA (cssDNA) production system that does not contain a non- endogenous antibiotic resistance gene, comprising: a production strain; and a phagemid, wherein the phagemid comprises a packaging signal, a designed sequence, and at least one selectable sequence encoding one or more of an auxotrophic marker, an antitoxin, an RNA that inhibits the expression of a gene that would retard or stop growth of the bacterium if it were expressed in the absence of the RNA, a transcription factor repressor that inhibits the expression of a gene that would retard or stop growth of the bacterium if it were expressed in the absence of the transcription factor repressor, a transcriptional activator that activates a transcriptional repressor, a sequence that expresses a tRNA that associates with an unnatural amino acid that the production strain is engineered to require, and combinations thereof.

4. The production system of claim 3, wherein the production strain genome comprises at least two phage protein coding sequences integrated into the production strain genome.

5. The production strain or system according to any of claims 1-4, wherein the production strain is susceptible to infection by single-stranded, filamentous bacteriophages of the realm of Monodnaviria, and furthermore is a Gram negative bacteria of a family selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae, Spirillaceae, Xanthomonadaceae, Clostridium, and Propionibacterium.

6. The production strain or system according to claim 5, wherein the production strain is susceptible to infection by a single stranded, filamentous bacteriophage selected from the group consisting of Ff, Fd, F1 and M13.

7. The production strain or system according to claim 6, wherein the production strain is susceptible to infection by the M13 bacteriophage.

8. The production strain or system of any one of claims 1-7, wherein the production strain is a strain of E. coli.

9. The production strain or system according to any one of claims 1-2 or 4-8, wherein the at least two phage protein coding sequences are differentially expressed as compared to each other.

10. The production strain or system according to any one of claims 1-2 or 4-8, wherein the at least two phage protein coding sequences are differentially expressed as compared to the native phage genome expression.

11. The production strain or system according to claim 9 or claim 10, wherein the at least two phage protein coding sequences encode at least two phage proteins selected from the group consisting of: p1, p2, p8, p10, and p11.

12. The production strain or system according to claim 11, wherein at least one of the at least two phage proteins are selected from the group consisting of: p3 and p5.

13. The production strain or system according to claim 12, wherein p3 activity is decreased as compared to native phage genome activity.

14. The production strain or system according to claim 12, wherein p5 activity is decreased as compared to native phage genome activity.

15. The production strain or system according to claim 1 or claim 2, wherein the at least two phage protein coding sequences are encoded by a filamentous bacteriophage of the realm of Monodnaviria.

16. The production strain or system of claim 15, wherein the filamentous bacteriophage is selected from a group consisting of: M13, Ff, Fd, Enterobacteria phage F1 [EF068134], Enterobacteria phage ID2, Enterobacteria phage NL95 [AF059243], Enterobacteria phage SP [X07489], Enterobacteria phage TW28, Enterobacteria phage Qbeta, Enterobacteria phage Qβ [AY099114], Enterobacteria phage M11 [AF059242], Enterobacteria phage ST, Enterobacteria phage TW18 [FJ483840], and Enterobacteria phage VK or a functional equivalent thereof.

17. The production strain or system according to claim 15 or claim 16, wherein the at least two phage protein coding sequences comprise at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 phage protein coding sequences.

18. The production strain or system according to claim 1 or claim 2, wherein the at least two phage protein coding sequences include sequences encoding one or more bacteriophage M13 proteins selected from the group consisting of p1, p2, p3, p4, p5, p6, p7, p8, p9, p10 and p11.

19. The production strain or system according to claim 18, wherein the bacteriophage M13 protein sequences include M13 bacteriophage genes selected from the group consisting of I, II, III, IV, V, VI, VII, VIII, IX, X and XI.

20. The production strain or system according to claim 1 or claim 2, wherein the at least two phage protein coding sequences comprise at least three phage protein coding sequences.

21. The cssDNA production system according to claim 3, wherein the production strain comprises at least two genomically integrated phage protein coding sequences.

22. The cssDNA production system according to claim 21, wherein the at least two genomically integrated phage protein coding sequences are selected from sequences from one or more filamentous bacteriophages of the realm of Monodnaviria.

23. The cssDNA production system according to claim 22, wherein the one or more filamentous bacteriophages are selected from a group consisting of: M13, Ff, Fd, Enterobacteria phage F1 [EF068134], Enterobacteria phage ID2, Enterobacteria phage NL95 [AF059243], Enterobacteria phage SP [X07489], Enterobacteria phage TW28, Enterobacteria phage Qbeta, Enterobacteria phage Qβ [AY099114], Enterobacteria phage M11 [AF059242], Enterobacteria phage ST, Enterobacteria phage TW18 [FJ483840], Enterobacteria phage VK, and functional equivalents thereof.

24. The cssDNA production system according to claim 3, wherein the selectable sequence is an antitoxin sequence from a toxin / antitoxin system.

25. The ccsDNA production system according to claim 24, wherein the toxin / antitoxin system is selected from a group consisting of ccdB / ccdA, hokA / sokA, pemK / pemI, mazF / mazE, ChpBK ChpBI, relE / relB, parE / parD, hipA / hipB, and other toxin / antitoxin systems where the toxin is expressed from the host genome and the antitoxin is expressed from the phagemid.

26. The cssDNA production system according to claim 3, wherein the selectable sequence produces at least one RNA molecule that down regulates the expression of a counterselectable sequence selected from the group consisting of HSVtk, Ura3, tetA, sacB, rpsL, pheS, pheS*, pheS**, thyA, lacY, gata-1, ccdB, hokA, pemK, mazF, chpBK, relE, parE, hipA and other toxins.

27. The cssDNA production system according to claim 3, wherein the selectable sequence encodes a transcriptional factor repressor.

28. The cssDNA production system according to claim 27, wherein the transcriptional factor repressor is selected from the group consisting of tetR, araC, lacI, xylS, and other sequences that reduce the expression of a counterselectable marker or toxin.

29. The cssDNA production system according to claim 3, wherein the transcriptional activator is selected from the group consisting of araC and xylR.

30. The cssDNA production system according to claim 3, wherein the auxotrophic marker is selected from the group consisting of uracil adenine, cytosine, guanine, thymine, alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, proline, Serine, threonine, Tryptophan, tyrosine, valine, biotin, uridine-5′- monophosphate, pantothenate, xanthine, spermidine, para aminobenzoate, lipoate, nicotinamide riboside, nicotinamide mononucleotide, D-glucosamine, thiamin, shikimate, aminoethyl-phosphonate, beta alanine, s-methyl-metyhionine, ornithine, indole, indole acetic acid, L-threonine, L-threonine O- 3-phosphate, nicotinate, ribosylnicotinamide, pyrimidine, agmatine, purine, and genes for the synthesis of other essential compounds expressed from the phagemid to complement a naturally occurring absence or synthetic deletion of a gene of the same or similar function in the host genome.

31. The production strain according to claim 1, further comprising the phagemid, wherein the phagemid comprises a packaging signal, a designed sequence, and at least one selectable sequence, optionally wherein the selectable sequence is selected from the group consisting of a sequence encoding an auxotrophic marker, an antitoxin, an RNA that inhibits the expression of a gene thatwould retard or stop growth of the bacterium if it were expressed in the absence of the RNA, a transcription factor repressor that inhibits the expression of a gene that would retard or stop growth of the bacterium if it were expressed in the absence of the transcription factor, a transcriptional activator that activates such a repressor, or a sequence that expresses a tRNA that associates with an unnatural amino acid that the production strain is engineered to require.

32. The production strain according to claim 31, wherein the auxotrophic marker is selected from the group consisting of uracil adenine, cytosine, guanine, thymine, alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, proline, Serine, threonine, Tryptophan, tyrosine, valine, biotin, uridine-5′- monophosphate, pantothenate, xanthine, spermidine, para aminobenzoate, lipoate, nicotinamide riboside, nicotinamide mononucleotide, D-glucosamine, thiamin, shikimate, aminoethyl-phosphonate, beta alanine, s-methyl-metyhionine, ornithine, indole, indole acetic acid, L-threonine, L-threonine O- 3-phosphate, nicotinate, ribosylnicotinamide, pyrimidine, agmatine, purine, and genes for the synthesis of other essential compounds expressed from the phagemid to complement a naturally occurring absence or synthetic deletion of a gene of the same or similar function in the host genome.

33. The production strain or system according to claim 1 or claim 2, wherein the at least two phage protein coding sequences comprise at least two phage genes, wherein the at least two phage genes are operably linked to synthetic promoters to optimize expression for cssDNA production comprising one or more of canonical T7 promoters and mutant T7 promoters under the control of an inducible T7 polymerase, lacI, lacIq, araBAD, tet, temperature sensitive promoters, stress responsive promoters, quorums sensing promoters, light sensitive promoters and other inducible or repressible promoters.

34. The production strain or system according to claim 1 or claim 2, wherein the at least two phage protein coding sequences are integrated into the production strain genome at least two distinct loci.

35. The production strain or system according to claim 1 or claim 2, wherein at least one of the at least two phage protein coding sequences are altered from their endogenous sequence through random mutagenesis, rational design, assisted lab evolution, directed evolution or combinations thereof to create proteins that produce higher yields or more pure yields of cssDNA.

36. The cssDNA production system according to claim 2, wherein the phagemid expresses the tRNA necessary to translate a recoded codon of an unnatural amino acid, wherein the production strain cannot incorporate the unnatural amino acid into its proteins without the presence of the phagemid, and neither the phagemid nor the production strain can multiply or replicate without the presence of the unnatural amino acid.

37. The production strain or system according to claim 1 or claim 2, wherein at least one of the at least two phage protein coding sequences additionally comprises a tag.

38. The production strain according to claim 37, wherein the tag is selected from an affinity tag or a detection tag.

39. The production strain according to claim 38, wherein the detection tag is selected from a group consisting of: a fluorescent tag, a luminescent tag, a chromophoric tag, and another tag to enable rapid quantification of the number of phage particles in solution.

40. The production strain according to claim 38, wherein the affinity tag is selected from a group consisting of: biotin, his, myc, flag, CBP, GST, HA, HBH, MBP, S, V5 and another affinity tag to aid in purification of phage particles from production broth.

41. The production strain or system of any of claims 1-40, wherein the phagemid comprises a pUC origin of replication or a derivative thereof.

42. The production strain or system of any of claims 1-40, wherein the phagemid comprises a pST19, pDHA29, pDHA30, pDHK29, pDHK30, or runaway R1 origin of replication or derivative thereof.

43. The production strain or system of any of claims 1-42, wherein the phagemid comprises an inc1 mutation and / or an inc2 mutation, optionally wherein the phagemid comprises an inc1 mutation and an inc2 mutation.

44. The production strain or system of any of claims 1-43, wherein the phagemid comprises an inc3 mutation.

45. The production strain or system of any of claims 1-44, wherein the phagemid comprises an inc5 mutation.

46. The production strain or system of any of claims 1-45, wherein the phagemid copy number in production strain cells grown to late log phase is at least 1,000, optionally wherein the phagemid copy number in production strain cells grown to late log phase is at least 2,000, at least 4,000, at least 7,000, at least 8,000, or at least 15,000.

47. A circular single-stranded DNA (cssDNA) production system, comprising: a production strain; and a phagemid, wherein the phagemid comprises a packaging signal, a designed sequence, and at least one selectable sequence, wherein the phagemid comprises a pUC origin of replication or a derivative thereof, and wherein the phagemid comprises an inc1 mutation and / or an inc2 mutation, optionally wherein the phagemid comprises an inc1 mutation and an inc2 mutation.

48. The production system of claim 47, wherein the production strain comprises at least two phage protein coding sequences integrated into the production strain genome.

49. A method of producing cssDNA, comprising; culturing a production strain according to any of claims 1, 5-20, 31-35, or 37-46, in media; introducing the phagemid; and collecting the phage particles; andseparating the cssDNA.

50. The method according to claim 49, further comprising altering at least one of the at least two phage protein coding sequences.

51. The method of claim 50, wherein the method further comprises comparing the collected cssDNA to cssDNA collected from a parental strain from which the production strain was made to determine if the altered sequence improves titer or quality of cssDNA produced.

52. The method according to claim 49, wherein the production strain further comprises at least one phage protein comprising a tag.

53. The method according to claim 52, wherein the tag is used to separate the phage.

54. A method of producing cssDNA, comprising; culturing cssDNA production system according to any of claims 2-48, in media; collecting the phage particles; and separating the cssDNA.

55. The method according to claim 54, wherein the production strain further comprises at least one phage protein comprising a tag.

56. The method according to claim 55, wherein the tag is used to separate the phage.

57. The production system of claim 3 or the method of claim 54, wherein the production strain comprises a helper plasmid derived from helper phage M13KO7 by removing the F1 origin and the packaging signal, optionally wherein the helper plasmid copy number in production strain cells grown to late log phase is at least 1,000, optionally wherein the helper plasmid copy number in production strain cells grown to late log phase is at least 2,000, at least 4,000, at least 7,000, at least 8,000, or at least 15,000.

58. The production strain, system or method of claim 57, wherein the helper plasmid comprises a pUC origin of replication.

59. The production strain, system, or method of any preceding claim, wherein the production strain is an engineered variant of a BW25113 strain.

60. The production strain, system or method according to any of claims 33-59, wherein a nucleotide sequence encoding the T7 polymerase is integrated into the production strain genome at an endA locus.

61. The production strain, system or method of claim 60, wherein the nucleotide sequence encoding the T7 polymerase is under control of an IPTG-inducible lac promoter.

62. The production strain, system, or method of any preceding claim, wherein the production strain comprises M13 genes II, V, VII, VIII, and IX integrated into the production strain genome at an intA locus.

63. The production strain, system, or method of any preceding claim, wherein the production strain comprises M13 genes III, VI, I, and IV integrated into the production strain genome at an intZ locus.

64. The production strain, system, or method of any preceding claim, further comprising M13 gene II integrated into the production strain genome at a mazF locus.

65. The production strain, system, or method of any preceding claim, further comprising M13 gene VIII integrated into the production strain genome at a mazF locus.

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