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1. (WO2018226893) A HIGH-THROUGHPUT (HTP) GENOMIC ENGINEERING PLATFORM FOR IMPROVING SACCHAROPOLYSPORA SPINOSA
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Claims

What is claimed is:

1. A high-throughput (HTP) method of genomic engineering to evolve a Saccharopolyspora sp. microbe to acquire a desired phenotype, comprising:

a. perturbing the genomes of an initial plurality of Saccharopolyspora microbes having the same genomic strain background, to thereby create an initial HTP genetic design Saccharopolyspora strain library comprising individual Saccharopolyspora strains with unique genetic variations;

b. screening and selecting individual Saccharopolyspora strains of the initial HTP genetic design Saccharopolyspora strain library for the desired phenotype;

c. providing a subsequent plurality of Saccharopolyspora microbes that each comprise a unique combination of genetic variation, said genetic variation selected from the genetic variation present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent HTP genetic design Saccharopolyspora strain library;

d. screening and selecting individual Saccharopolyspora strains of the subsequent HTP genetic design Saccharopolyspora strain library for the desired phenotype; and

e. repeating steps c)-d) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora microbe has acquired the desired phenotype, wherein each subsequent iteration creates a new HTP genetic design Saccharopolyspora strain library comprising individual Saccharopolyspora strains harboring unique genetic variations that are a combination of genetic variation selected from amongst at least two individual Saccharopolyspora strains of a preceding HTP genetic design Saccharopolyspora strain library.

2. The HTP method of genomic engineering according to claim 1 , wherein the function and/or identity of the genes that contain the genetic variations are not considered before the genetic variations are combined in step (b).

3. The HTP method of genomic engineering according to claim 1 , wherein at least one genetic variation to be combined is not in a genomic region that contains repeating segments of encoding DNA modules.

4. The HTP method of genomic engineering according to claim 1, wherein the subsequent plurality of Saccharopolyspora microbes that each comprises a unique combination of genetic variations in step (c) are produced by:

1) introducing a plasmid into an individual Saccharopolyspora strain belonging to the initial HTP genetic design Saccharopolyspora strain library, wherein the plasmid comprises a selection marker, a counterselection marker, a DNA fragment having homology to the genomic locus of the base Saccharopolyspora strain, and plasmid backbone sequence, wherein the DNA fragment has a genetic variation derived from another individual Saccharopolyspora strain also belonging to the initial HTP genetic design Saccharopolyspora strain library;

2) selecting for Saccharopolyspora strains with integration event based on the presence of the selection marker in the genome;

3) selecting for Saccharopolyspora strains having the plasmid backbone looped out based on the absence of the counterselection marker gene.

5. The HTP method of claim 4, wherein the plasmid does not comprise a temperature sensitive replicon.

6. The HTP method of claim 4, wherein the selection step (3) is performed without replication of the integrated plasmid.

7. The HTP method of genomic engineering according to claim 1 , wherein the initial HTP genetic design Saccharopolyspora strain library comprises at least one library selected from the group consisting of a promoter swap microbial strain library, SNP swap microbial strain library, start/stop codon microbial strain library, optimized sequence microbial strain library, a terminator swap microbial strain library, a transposon mutagenesis microbial strain diversity library, a ribosomal binding site microbial strain library, an anti-metabolite/fermentation product resistance library, a termination insertion microbial strain library, and any combination thereof.

8. The HTP method of genomic engineering according to claim 1, wherein the subsequent HTP genetic design Saccharopolyspora strain library is a full combinatorial Saccharopolyspora strain library of the initial HTP genetic design microbial strain library.

9. The HTP method of genomic engineering according to claim 1, wherein the subsequent HTP genetic design Saccharopolyspora strain library is a subset of a full combinatorial Saccharopolyspora strain library derived from the genetic variations in the initial HTP genetic design Saccharopolyspora strain library.

10. The HTP method of genomic engineering according to claim 1, wherein the subsequent HTP genetic design derived from the genetic variations in strain library is a full combinatorial microbial strain library derived from the genetic variations in a preceding HTP genetic design Saccharopolyspora strain library.

11. The HTP method of genomic engineering according to claim 1, wherein the subsequent HTP genetic design Saccharopolyspora strain library is a subset of a full combinatorial Saccharopolyspora strain library derived from the genetic variations in a preceding HTP genetic design Saccharopolyspora strain library.

12. The HTP method of genomic engineering according to claim 1, wherein perturbing the genome comprises utilizing at least one method selected from the group consisting of: random mutagenesis, targeted sequence insertions, targeted sequence deletions, targeted sequence replacements, transposon mutagenesis, and any combination thereof.

13. The HTP method of genomic engineering according to claim 1 , wherein the initial plurality of Saccharopolyspora microbes comprise unique genetic variations derived from a production Saccharopolyspora strain.

14. The HTP method of genomic engineering according to claim 1 , wherein the initial plurality of Saccharopolyspora microbes comprise production strain microbes denoted SiGeni and any number of subsequent microbial generations derived therefrom denoted SnGenn.

15. The HTP method of genomic engineering according to claim 1, wherein the step c comprises rapidly consolidating the genetic variations by using protoplast fusion techniques.

16. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a promoter swap microbial strain library.

17. The HTP method of genomic engineering according to claim 16, wherein the promoter swap microbial strain library comprises at least one promoter with a nucleotide sequence selected from SEQ ID Nos. 1 to 69 and 172 to 175.

18. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a SNP swap microbial strain library.

19. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a terminator swap microbial strain library.

20. The HTP method of genomic engineering according to claim 19, wherein the terminator swap microbial strain library comprises at least one terminator with a nucleotide sequence selected from SEQ ID Nos. 70 to 80.

21. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a transposon mutagenesis microbial strain diversity library.

22. The HTP method of genomic engineering according to claim 21, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a Loss-of -Function (LoF) transposon and/or a Gain-of-Function (GoF) transposon.

23. The HTP method of genomic engineering according to claim 22, wherein the GoF transposon comprises a solubility tag, a promoter, and/or a counter-selection marker.

24. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises a ribosomal binding site microbial strain library.

25. The HTP method of genomic engineering according to claim 24, wherein ribosomal binding site microbial strain library comprises at least one ribosomal binding site (RBS) with a nucleotide sequence selected from SEQ ID Nos. 97 to 127.

26. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design Saccharopolyspora strain library or the subsequent HTP genetic design Saccharopolyspora strain library comprises an anti-metabolite/fermentation product resistance library.

27. The HTP method of genomic engineering according to claim 26, wherein the anti-metabolite/fermentation product resistance library comprises a Saccharopolyspora strain resistance to a molecule involved in spinosyn synthesis in Saccharopolyspora.

28. A method for generating a SNP swap Saccharopolyspora strain library, comprising the steps of:

a. providing a reference Saccharopolyspora strain and a second Saccharopolyspora strain, wherein the second Saccharopolyspora strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, which are not present in the reference Saccharopolyspora strain; and

b. perturbing the genome of either the reference Saccharopolyspora strain, or the second Saccharopolyspora strain, to thereby create an initial SNP swap Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations corresponds to a single genetic variation selected from the plurality of identified genetic variations between the reference Saccharopolyspora strain and the second Saccharopolyspora strain.

29. The method for generating a SNP swap Saccharopolyspora strain library according to claim 28, wherein the genome of the reference Saccharopolyspora strain is perturbed to add one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are found in the second Saccharopolyspora strain.

30. The method for generating a SNP swap Saccharopolyspora strain library according to claim 28, wherein the genome of the second Saccharopolyspora strain is perturbed to remove one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are not found in the reference Saccharopolyspora strain.

31. The method for generating a SNP swap Saccharopolyspora strain library according to claim 28, wherein the resultant plurality of individual Saccharopolyspora strains with unique genetic variations, together comprise a full combinatorial library of all the identified genetic variations between the reference Saccharopolyspora strain and the second Saccharopolyspora strain.

32. The method for generating a SNP swap Saccharopolyspora strain library according to claim 28, wherein the resultant plurality of individual Saccharopolyspora strains with unique genetic variations, together comprise a subset of a full combinatorial library of all the identified genetic variations between the reference Saccharopolyspora strain and the second Saccharopolyspora strain.

33. A method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a. providing a parental lineage Saccharopolyspora strain and a production Saccharopolyspora strain derived therefrom, wherein the production Saccharopolyspora strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, not present in the parental lineage Saccharopolyspora strain;

b. perturbing the genome of either the parental lineage Saccharopolyspora strain, or the production Saccharopolyspora strain, to thereby create an initial Saccharopolyspora strain library. Wherein each strain in the initial library comprises a unique genetic variation from the plurality of identified genetic variations between the parental lineage Saccharopolyspora strain and the production Saccharopolyspora strain;

c. screening and selecting individual Saccharopolyspora strains of the initial SNP swap Saccharopolyspora strain library for phenotype performance improvements over a reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

d. providing a subsequent plurality of microbes that each comprise a combination of unique genetic variation from the variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent library of Saccharopolyspora strains;

e. screening and selecting individual strains of the subsequent strain library for phenotypic performance improvements over the reference Saccharopolyspora

strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new library of Saccharopolyspora strains„ where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding library.

34. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the initial library of Saccharopolyspora strains is a full combinatorial library comprising all of the identified genetic variations between the parental lineage Saccharopolyspora strain and the production Saccharopolyspora strain.

35. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the initial library of Saccharopolyspora strains is a subset of a full combinatorial library comprising a subset of the identified genetic variations between the reference parental lineage Saccharopolyspora strain and the production Saccharopolyspora strain.

36. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the subsequent library of Saccharopolyspora strains is a full combinatorial library of the initial library.

37. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the subsequent library of Saccharopolyspora strains is a full combinatorial library of the initial library.

38. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the subsequent library of Saccharopolyspora strains is a full combinatorial library of a preceding library.

39. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the subsequent library of Saccharopolyspora strains is a subset of a full combinatorial library of a preceding library.

40. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the genome of the parental lineage Saccharopolyspora strain is perturbed to add one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are found in the production Saccharopolyspora strain.

41. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the genome of the production Saccharopolyspora strain is perturbed to remove one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are not found in the parental lineage Saccharopolyspora strain.

42. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein perturbing the genome comprises utilizing at least one method selected from the group consisting of: random mutagenesis, targeted sequence insertions, targeted sequence deletions, targeted sequence replacements, and combinations thereof.

43. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

44. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

45. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the improved phenotypic performance of step f) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

46. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the improved phenotypic performance of step f) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

47. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 46, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

48. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 46, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

49. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the identified genetic variations further comprise artificial promoter swap genetic variations from a promoter swap library.

50. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, further comprising engineering the genome of at least one microbial strain of either the initial library of Saccharopolyspora strains, or a subsequent library of Saccharopolyspora strains, to comprise one or more promoters from a promoter ladder operably linked to an endogenous Saccharopolyspora target gene.

51. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 33, wherein the strain library comprises at least one library selected from the group consisting of a promoter swap microbial strain library, SNP swap microbial strain library, start/stop codon microbial strain library, optimized sequence microbial strain library, a terminator swap microbial strain library, a transposon mutagenesis microbial strain diversity library, a ribosomal binding site microbial strain library, an anti-metabolite/fermentation product resistance library, a termination insertion microbial strain library, and any combination thereof.

52. The method for rehabilitating and improving the phenotypic performance of a production Saccharopolyspora strain according to claim 51 , wherein the strain library comprises at least one library selected from the group consisting of:

1) a promoter swap microbial strain library comprising at least one promoter having a sequence selected from SEQ ID No. 1-69;

2) a terminator swap microbial strain library comprising at least one terminator having a sequence selected from SEQ ID Nos. 70 to 80; and

3) a ribosomal binding site (RBS) library comprising at least one RBS having a sequence selected from SEQ ID Nos. 97 to 127.

53. A method for generating a promoter swap Saccharopolyspora strain library, said method comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a promoter ladder, wherein said promoter ladder comprises a plurality of promoters exhibiting different expression profiles in the base Saccharopolyspora strain; and

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial promoter swap Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the promoters from the promoter ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain.

54. The method for generating a promoter swap Saccharopolyspora strain library according to claim 53, wherein at least one of the plurality of promoters comprises a promoter having a sequence selected from SEQ ID No. 1-69.

55. A promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a promoter ladder, wherein said promoter ladder comprises a plurality of promoters exhibiting different expression profiles in the base Saccharopolyspora strain;

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial promoter swap Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the promoters from the promoter ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain;

c. screening and selecting individual Saccharopolyspora strains of the initial promoter swap Saccharopolyspora strain library for phenotypic performance improvements over a reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer the phenotypic performance improvements; d. providing a subsequent plurality of Saccharopolyspora microbes that each comprise a combination of unique genetic variations from the genetic variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent promoter swap Saccharopolyspora strain library;

e. screening and selecting individual Saccharopolyspora strains of the subsequent promoter swap Saccharopolyspora strain library for the desired phenotypic performance improvements over the reference E. coli strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new promoter swap Saccharopolyspora strain library of Saccharopolyspora strains, wherein each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding promoter swap Saccharopolyspora strain library.

56. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the subsequent promoter swap Saccharopolyspora strain library is a full combinatorial library of the initial promoter swap Saccharopolyspora strain library.

57. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the subsequent promoter swap Saccharopolyspora strain library is a full combinatorial library of the initial promoter swap Saccharopolyspora strain library.

58. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the subsequent promoter swap Saccharopolyspora strain library is a subset of a full combinatorial library of the initial promoter swap Saccharopolyspora strain library.

59. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the subsequent promoter swap Saccharopolyspora strain library is a full combinatorial library of a preceding promoter swap Saccharopolyspora strain library.

60. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the subsequent promoter swap Saccharopolyspora strain library is a subset of a full combinatorial library of a preceding promoter swap Saccharopolyspora strain library.

61. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein steps d)-e) are repeated until the phenotypic performance a Saccharopolyspora strain of a subsequent promoter swap Saccharopolyspora strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

62. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent promoter swap Saccharopolyspora strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

63. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the improved phenotypic performance of step f) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

64. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the improved phenotypic performance of step f) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

65. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 64, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

66. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 65, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

67. The promoter swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 55, wherein the promoter ladder comprises at least one promoter with a nucleotide sequence selected from SEQ ID No. 1 - 69.

68. A method for generating a terminator swap Saccharopolyspora strain library, comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a terminator ladder, wherein said terminator ladder comprises a plurality of terminators exhibiting different expression profiles in the base Saccharopolyspora strain; and

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial terminator swap Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the terminators from the terminator ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain.

69. A terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a terminator ladder, wherein said terminator ladder comprises a plurality of terminators exhibiting different expression profiles in the base Saccharopolyspora strain;

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial terminator swap Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the terminators from the terminator ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain;

c. screening and selecting individual Saccharopolyspora strains of the initial terminator swap Saccharopolyspora strain library for phenotypic performance improvements over a reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

d. providing a subsequent plurality of Saccharopolyspora microbes that each comprise a combination of unique genetic variations from the genetic variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent terminator swap Saccharopolyspora strain library;

e. screening and selecting individual Saccharopolyspora strains of the subsequent terminator swap Saccharopolyspora strain library for phenotypic performance improvements over the reference Saccharopolyspora strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new terminator swap Saccharopolyspora strain library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding library.

70. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the subsequent terminator swap Saccharopolyspora strain library is a full combinatorial library of the initial terminator swap Saccharopolyspora strain library.

71. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the subsequent terminator swap Saccharopolyspora strain library is a subset of a full combinatorial library of the initial terminator swap Saccharopolyspora strain library.

72. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the subsequent terminator swap

Saccharopolyspora strain library is a full combinatorial library of a preceding terminator swap Saccharopolyspora strain library.

73. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the subsequent terminator swap Saccharopolyspora strain library is a subset of a full combinatorial library of a preceding terminator swap Saccharopolyspora strain library.

74. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent terminator swap Saccharopolyspora strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

75. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent terminator swap Saccharopolyspora strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

76. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the improved phenotypic performance of step f) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

77. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the improved phenotypic performance of step f) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic

acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

78. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 77, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

79. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 78, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

80. The terminator swap method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 69, wherein the terminator ladder comprises at least one terminator with a nucleotide sequence selected from SEQ ID No. 70 - 80.

81. A method for generating a ribosomal binding site (RBS) Saccharopolyspora strain library, comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a RBS ladder, wherein said RBS ladder comprises a plurality of RBSs exhibiting different expression profiles in the base Saccharopolyspora strain; and

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial RBS Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the RBSs from the RBS ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain.

82. A method for improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a. providing a plurality of target genes endogenous to a base Saccharopolyspora strain, and a RBS ladder, wherein said RBS ladder comprises a plurality of RBSs exhibiting different expression profiles in the base Saccharopolyspora strain;

b. engineering the genome of the base Saccharopolyspora strain, to thereby create an initial RBS Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of the RBSs from the RBS ladder operably linked to one of the target genes endogenous to the base Saccharopolyspora strain;

c. screening and selecting individual Saccharopolyspora strains of the initial RBS Saccharopolyspora strain library for phenotypic performance improvements over a reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

d. providing a subsequent plurality of Saccharopolyspora strains that each comprise a combination of unique genetic variations from the genetic variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent RBS Saccharopolyspora strain library;

e. screening and selecting individual Saccharopolyspora strains of the subsequent RBS Saccharopolyspora strain library for phenotypic performance improvements over the reference Saccharopolyspora strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new RBS Saccharopolyspora strain library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding library.

83. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the subsequent RBS Saccharopolyspora strain library is a full combinatorial library of the initial RBS Saccharopolyspora strain library.

84. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the subsequent RBS Saccharopolyspora strain library is a subset of a full combinatorial library of the initial RBS Saccharopolyspora strain library.

85. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the subsequent RBS Saccharopolyspora strain library is a full combinatorial library of a preceding RBS Saccharopolyspora strain library.

86. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the subsequent RBS Saccharopolyspora strain library is a subset of a full combinatorial library of a preceding RBS Saccharopolyspora strain library.

87. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent RBS Saccharopolyspora strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

88. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein steps d)-e) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent RBS Saccharopolyspora strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

89. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the improved phenotypic performance of step f) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

90. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the improved phenotypic performance of step f) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

91. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 90, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

92. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 91, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

93. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 82, wherein the RBS ladder comprises at least one RBS with a nucleotide sequence selected from SEQ ID No. 97-127.

94. A method for generating a transposon mutagenesis Saccharopolyspora strain diversity library, comprising

a) introducing a transposon into a population of cells of one or more base Saccharopolyspora strains; and

b) selecting for Saccharopolyspora strain comprising randomly integrated transposon, thereby creating an initial Saccharopolyspora strain library comprising a plurality of individual

Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more randomly integrated transposon.

95. The method of claim 94, further comprising:

c). selecting for a subsequence Saccharopolyspora strain library exhibits at least one increase in a measured phenotypic variable compared to the phenotypic performance of the base Saccharopolyspora strain.

96. The method of claim 94, wherein the transposon is introduced into the base Saccharopolyspora strain using a complex of transposon and transposase protein which allows for in vivo transposition of the transposon into the genome of the Saccharopolyspora strain.

97. The method of claim 94, wherein the transposase protein is derived from EZ-Tn5 transposome system.

98. The method of claim 94, wherein the transposon is a Loss-of-Function (LoF) transposon, or a Gain-of-Function (GoF) transposon.

99. The method of claim 94, wherein the GoF transposon comprises a solubility tag, a promoter, and/or a counter-selection marker.

100. A method for improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a. engineering the genome of a base Saccharopolyspora strain by transposon mutagenesis, to thereby create an initial transposon mutagenesis Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more transposon;

b. screening and selecting individual Saccharopolyspora strains of the initial transposon mutagenesis Saccharopolyspora strain library for phenotypic performance improvements over a

reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

c. providing a subsequent plurality of Saccharopolyspora strains that each comprise a combination of unique genetic variations from the genetic variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent transposon mutagenesis Saccharopolyspora strain library;

d. screening and selecting individual Saccharopolyspora strains of the subsequent transposon mutagenesis Saccharopolyspora strain library for phenotypic performance improvements over the reference Saccharopolyspora strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

e. repeating steps c)-d) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new transposon mutagenesis Saccharopolyspora strain library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding library.

101. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the subsequent transposon mutagenesis Saccharopolyspora strain library is a full combinatorial library of the initial transposon mutagenesis Saccharopolyspora strain library.

102. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the subsequent transposon mutagenesis Saccharopolyspora strain library is a subset of a full combinatorial library of the initial transposon mutagenesis Saccharopolyspora strain library.

103. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the subsequent transposon mutagenesis Saccharopolyspora strain library is a full combinatorial library of a preceding transposon mutagenesis Saccharopolyspora strain library.

104. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the subsequent transposon mutagenesis Saccharopolyspora strain library is a subset of a full combinatorial library of a preceding transposon mutagenesis Saccharopolyspora strain library.

105. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein steps c)-d) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent transposon mutagenesis Saccharopolyspora strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

106. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein steps c)-d) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent transposon mutagenesis Saccharopolyspora strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

107. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the improved phenotypic performance of step e) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

108. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the improved phenotypic performance of step e) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic acid,

synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

109. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 108, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

110. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 109, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

111. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 100, wherein the transposon comprises is a Loss-of-F unction (LoF) transposon, or a Gain-of-Function (GoF) transposon.

112. The method of claim 111 , wherein the GoF transposon comprises a solubility tag, a promoter, and/or a counter-selection marker.

113. A method for generating an anti-metabolite/fermentation product resistant Saccharopolyspora strain library, comprising the step of:

a) selecting for Saccharopolyspora strains resistant to a predetermined metabolite and/or a fermentation product, thereby creating an initial Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein at least one of said unique genetic variations results in resistance to the predetermined metabolite and/or a fermentation product; and

b) collecting Saccharopolyspora strains resistant to the predetermined metabolite and/or the fermentation product to generate the anti-metabolite/fermentation product resistant Saccharopolyspora strain library.

114. The method for generating an anti-metabolite/fermentation product resistant Saccharopolyspora strain library of claim 113, wherein the predetermined metabolite and/or fermentation product is selected from the group consisting of molecules involved in the spinosyn synthesis pathway, molecules involved in the SAM/methionine pathway, molecules involved in the lysine production pathway, molecules involved in the tryptophan pathway, molecules involved in the threonine pathway, molecules involved in the acetyl-CoA production pathway, and molecules involved in the de-novo or salvage purine and pyrimidine pathways.

115. The method for generating an anti-metabolite/fermentation product resistant Saccharopolyspora strain library of claim 114, wherein:

1) the molecule involved in the spinosyn synthesis pathway is a spinosyn, and optionally each strain is resistant to about 50 ug/ml to about 2 mg/ml spinosyn J/L;

2) the molecule involved in the SAM/methionine pathway is alpha-methyl methionine (aMM) or norleucine, and optionally each strain is resistant to about 1 mM to about 5 mM alpha-methyl methionine (aMM);

3) the molecule involved in the lysine production pathway is thialysine or a mixture of alpha-ketobytarate and aspartate hydoxymate;

4) the molecule involved in the tryptophan pathway is azaserine or 5-fuoroindole;

5) the molecule involved in the threonine pathway is beta-hydroxynorvaline;

6) the molecule involved in the acetyl-CoA production pathway is cerulenin, and

7) the molecule involved in the de-novo or salvage purine and pyrimidine pathways is a purine or a pyrimidine analog.

116. The method for generating an anti-metabolite/fermentation product resistant Saccharopolyspora strain library of claim 113, further comprising the step of:

b). selecting for a subsequence Saccharopolyspora strain library exhibits at least one increase in a measured phenotypic variable compared to the phenotypic performance of the base Saccharopolyspora strain.

117. The method for generating an anti-metabolite/fermentation product resistant Saccharopolyspora strain library of claim 116, wherein each strain in the subsequence Saccharopolyspora strain library exhibits an increased synthesis of a spinosyn.

118. A method for improving the phenotypic performance of a production Saccharopolyspora strain, comprising the steps of:

a) providing an initial anti-metabolite/fermentation product resistant Saccharopolyspora strain library comprising a plurality of individual Saccharopolyspora strains with unique genetic variations found within each strain of said plurality of individual Saccharopolyspora strains, wherein each of said unique genetic variations comprises one or more of genetic variations, wherein the genetic variations confer resistance to a predetermined metabolite or a fermentation product;

b) screening and selecting individual Saccharopolyspora strains of the initial anti-metabolite/fermentation product resistant Saccharopolyspora strain library for phenotypic performance improvements over a reference Saccharopolyspora strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

c) providing a subsequent plurality of Saccharopolyspora strains that each comprise a combination of unique genetic variations from the genetic variations present in at least two individual Saccharopolyspora strains screened in the preceding step, to thereby create a subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library;

d) screening and selecting individual Saccharopolyspora strains of the subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library for phenotypic performance improvements over the reference Saccharopolyspora strain, thereby identifying unique combinations of genetic variation that confer additional phenotypic performance improvements; and

e) repeating steps c)-d) one or more times, in a linear or non-linear fashion, until a Saccharopolyspora strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production Saccharopolyspora strain, wherein each subsequent iteration creates a new anti-metabolite/fermentation product resistant Saccharopolyspora strain library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual Saccharopolyspora strains of a preceding library.

119. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 118, wherein the subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library is a full combinatorial library of the initial anti-metabolite/fermentation product resistant Saccharopolyspora strain library.

120. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 118, wherein the subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library is a subset of a full combinatorial library of the initial anti-metabolite/fermentation product resistant Saccharopolyspora strain library.

121. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 118, wherein the subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library is a full combinatorial library of a preceding anti-metabolite/fermentation product resistant Saccharopolyspora strain library.

122. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 118, wherein the subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library is a subset of a full combinatorial library of a preceding anti-metabolite/fermentation product resistant Saccharopolyspora strain library.

123. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 1 18, wherein steps c)-d) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

124. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 1 18, wherein steps c)-d) are repeated until the phenotypic performance of a Saccharopolyspora strain of a subsequent anti-metabolite/fermentation product resistant Saccharopolyspora strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production Saccharopolyspora strain.

125. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 118, wherein the improved phenotypic performance of step e) is selected from the group consisting of: volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

126. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 125, wherein the improved phenotypic performance of step e) is: increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

127. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 126, wherein the product of interest is selected from the group consisting of a spinosyn, spinosad, spinetoram, genistein, choline oxidase, a coumamidine compound, erythromycin, ivermectin aglycone, a HMG-CoA reductase inhibitor, a carboxylic acid isomer, alpha-methyl methionine, thialysine, alpha-ketobytarate, aspartate hydoxymate, azaserine, 5-fuoroindole, beta-hydroxynorvaline, cerulenin, purine, pyrimidine, and analogs thereof.

128. The method for improving the phenotypic performance of a production Saccharopolyspora strain according to claim 127, wherein the spinosyn is spinosyn A, spinosyn D, spinosyn J, spinosyn L, or combinations thereof.

129. A Saccharopolyspora host cell comprising a promoter operably linked to an endogenous gene of the host cell, wherein the promoter is heterologous to the endogenous gene, wherein the promoter has a sequence selected from the group consisting of SEQ ID Nos. 1 - 69.

130. The Saccharopolyspora host cell of claim 129, wherein the endogenous gene is involved in synthesis of a spinosyn in the Saccharopolyspora host cell.

131. The Saccharopolyspora host cell of claim 129, wherein Saccharopolyspora host cell has a desired level of improved phenotypic performance compared to the phenotypic performance of a reference Saccharopolyspora strain without the promoter operably linked to the endogenous gene.

132. A Saccharopolyspora strain library, wherein each Saccharopolyspora strain in the library comprises a promoter operably linked to an endogenous gene of the host cell, wherein the promoter is heterologous to the endogenous gene, wherein the promoter has a sequence selected from the group consisting of SEQ ID Nos. 1 - 69.

133. A Saccharopolyspora host cell comprising a terminator linked to an endogenous gene of the host cell, wherein the terminator is heterologous to the endogenous gene, wherein the promoter has a sequence selected from the group consisting of SEQ ID Nos. 70-80.

134. The Saccharopolyspora host cell of claim 133, wherein the endogenous gene is involved in synthesis of a spinosyn in the Saccharopolyspora host cell.

135. The Saccharopolyspora host cell of claim 133, wherein Saccharopolyspora host cell has a desired level of improved phenotypic performance compared to the phenotypic performance of a reference Saccharopolyspora strain without the promoter operably linked to the endogenous gene.

136. A Saccharopolyspora strain library, wherein each Saccharopolyspora strain in the library comprises a terminator linked to an endogenous gene of the host cell, wherein the terminator is heterologous to the endogenous gene, wherein the terminator has a sequence selected from the group consisting of SEQ ID Nos. 70-80.

137. A Saccharopolyspora host cell comprising a ribosomal binding site operably linked to an endogenous gene of the host cell, wherein the ribosomal binding site is heterologous to the endogenous gene, wherein the ribosomal binding site has a sequence selected from the group consisting of SEQ ID Nos. 97 - 127.

138. The Saccharopolyspora host cell of claim 137, wherein the endogenous gene is involved in synthesis of a spinosyn in the Saccharopolyspora host cell.

139. The Saccharopolyspora host cell of claim 137, wherein Saccharopolyspora host cell has a desired level of improved phenotypic performance compared to the phenotypic performance of a reference Saccharopolyspora strain without the RBS operably linked to the endogenous gene.

140. A Saccharopolyspora strain library, wherein each Saccharopolyspora strain in the library comprises a ribosomal binding site operably linked to an endogenous gene of the host cell, wherein the ribosomal binding site is heterologous to the endogenous gene, wherein the ribosomal binding site has a sequence selected from the group consisting of SEQ ID Nos. 97 - 127.

141. A Saccharopolyspora host cell comprising a transposon, wherein Saccharopolyspora host cell has a desired level of improved phenotypic performance compared to the phenotypic performance of a reference Saccharopolyspora strain without the transposon.

142. The Saccharopolyspora host cell of claim 141, wherein the transposon is a Loss-of-Function (LoF) transposon, or a Gain-of-Function (GoF) transposon.

143. The Saccharopolyspora host cell of claim 142, wherein the Gain-of-Function (GoF) transposon comprises a promoter, a counterselection marker, and/or a solubility tag.

144. The Saccharopolyspora host cell of claim 141, wherein the transposon comprises a sequence selected from the group consisting of SEQ ID No. 128-131.

145. A Saccharopolyspora strain library, wherein each Saccharopolyspora strain in the library comprises a transposon having a sequence selected from the group consisting of SEQ ID No. 128-131, wherein the transposon in each strain is at a different genomic locus.

146. A Saccharopolyspora strain library, wherein each Saccharopolyspora strain in the library comprises a genetic variation that results in resistance of the strain to

1) a molecule involved in the spinosyn synthesis pathway,

2) a molecule involved in the S AM/methionine pathway,

3) a molecule involved in the lysine production pathway,

4) a molecule involved in the tryptophan pathway,

5) a molecule involved in the threonine pathway,

6) a molecule involved in the acetyl-CoA production pathway, and/or

7) a molecule involved in the de-novo or salvage purine and pyrimidine pathways.

147. The Saccharopolyspora strain library of claim 146, wherein:

1) the molecule involved in the spinosyn synthesis pathway is a spinosyn;

2) the molecule involved in the SAM/methionine pathway is alpha-methyl methionine (aMM) or norleucine;

3) the molecule involved in the lysine production pathway is thialysine or a mixture of alpha-ketobytarate and aspartate hydoxymate;

4) the molecule involved in the tryptophan pathway is azaserine or 5-fuoroindole;

5) the molecule involved in the threonine pathway is beta-hydroxynorvaline;

6) the molecule involved in the acetyl-CoA production pathway is cerulenin; and

7) the molecule involved in the de-novo or salvage purine and pyrimidine pathways is a purine or a pyrimidine analog.

148. The Saccharopolyspora strain library of claim 147, wherein the molecule is spinosyn J/L, and wherein each strain is resistant to about 50 ug/ml to about 2 mg/ml spinosyn J/L.

149. The Saccharopolyspora strain library of claim 147, wherein the molecule is alpha-methyl methionine (aMM), wherein each strain is resistant to about ImM to about 5mM aMM.

150. A Saccharopolyspora strain comprising a reporter gene, wherein the reporter gene is selected from the group consisting of:

a) genes encoding a green fluorescent reporter protein, optionally the genes are codon optimized for expression in Saccharopolyspora;

b) genes encoding a green fluorescent reporter protein, optionally the genes are codon optimized for expression in Saccharopolyspora; and

c) genes encoding a beta-glucuronidase (gusA) protein, optionally the genes are codon optimized for expression in Saccharopolyspora.

151. The Saccharopolyspora strain of claim 150, wherein:

a) the green fluorescent reporter protein has the amino acid sequence of SEQ ID No. 143;

b) the red fluorescent reporter protein has the amino acid sequence of SEQ ID No. 144; and c) the gusA protein has the amino acid sequence of SEQ ID No. 145.

152. The Saccharopolyspora strain of claim 150, wherein:

a) the gene encoding the green fluorescent reporter protein has the sequence of SEQ ID No. 81 ; b) the gene encoding the red fluorescent reporter protein has the sequence of SEQ ID No. 82; and c) the gene encoding the gusA protein has sequence of SEQ ID No. 83.

153. The Saccharopolyspora strain of claim 150, wherein the strain comprises both the gene encoding the green fluorescent reporter protein, and the gene encoding the red fluorescent reporter protein, wherein the fluorescent excitation and emission spectra of the green fluorescent reporter protein and the red fluorescent reporter protein are distinct from each other.

154. The Saccharopolyspora strain of claim 150, wherein the strain comprises both the gene encoding the green fluorescent reporter protein, and the gene encoding the red fluorescent reporter protein, wherein the fluorescent excitation and emission spectra of the green fluorescent reporter protein and the red fluorescent reporter protein are distinct from the endogenous fluorescence of the Saccharopolyspora strain.

155. A Saccharopolyspora strain comprising a DNA fragment integrated into one or more neutral integration sites in the genome of the Saccharopolyspora strain, wherein the neutral integration sites are selected from the group of positions within a genomic fragment having a sequence selected from SEQ ID Nos. 132 - 142, or genomic fragments homologous to any one of SEQ ID Nos. 132 - 142.

156. The Saccharopolyspora strain of claim 155, wherein the Saccharopolyspora strain has a desired level of improved phenotypic performance compared to the phenotypic performance of a reference Saccharopolyspora strain without the integrated DNA fragment.

157. The Saccharopolyspora strain of claim 156, wherein the Saccharopolyspora strain has a desired level of improved spinosyn production compared to the phenotypic performance of a reference Saccharopolyspora strain without the integrated DNA fragment.

158. The Saccharopolyspora strain of claim 155, wherein the integrated DNA fragment comprises a sequence encoding for a reporter protein.

159. The Saccharopolyspora strain of claim 155, wherein the integrated DNA fragment comprises a transposon.

160. The Saccharopolyspora strain of claim 155, wherein the integrated DNA fragment comprises an attachment site (attB) which can be recognized by its corresponding integrase.

161. A method of integrating a DNA fragment into the genome of a Saccharopolyspora strain, wherein the DNA fragment is integrated into a neutral integration site in the genome of the Saccharopolyspora strain, wherein the neutral integration site is selected from the group of positions within a genomic fragment having a sequence selected from SEQ ID Nos. 132 - 142, or genomic fragments homologous to any one of SEQ ID Nos. 132 - 142.

162. The method of integrating a DNA fragment into the genome of a Saccharopolyspora strain of claim 161, wherein the DNA fragment comprises an attachment site (attB) which can be recognized by its corresponding integrase.

163. A method for rapidly consolidating genetic mutations derived from at least two parental Saccharopolyspora strains, comprising the steps of:

(1) providing at least two parental Saccharopolyspora strains, wherein each strain comprises a unique genomic mutation that does not exist in the other strains;

(2) preparing protoplasts from each of the parental strains;

(3) fusing the protoplasts from the parental strains to produce fused protoplast comprising the genomes of two parental Saccharopolyspora strains, wherein homologous recombination between the genomes of each parental strain occurs;

(4) recovering Saccharopolyspora cells from the fused protoplast produced in step (3); and

(5) selecting for Saccharopolyspora cells comprising the unique genomic mutation of a first parental Saccharopolyspora strain; and

(6) genotyping the Saccharopolyspora cells obtained in step (5) for the presence of the unique genomic mutation of a second parental strain,

thereby obtaining a new Saccharopolyspora strain comprising the unique genomic mutations derived from two parental Saccharopolyspora strains.

164. The method of claim 163, wherein one of the unique genomic mutations is linked to a selectable marker, while the other unique genomic mutation is not linked to any selectable marker.

165. The method of claim 164, wherein in step (3) the ratio of protoplasts of the stain originally containing the unique genomic mutation linked to the selectable marker : protoplasts of the stain originally containing the unique genomic mutation not linked to the selectable marker is less than 1 : 1.

166. The method of claim 165, wherein the ratio is about 1 : 10 to about 1 : 100, or less.

167. The method of claim 163, wherein in step (4), protoplast cells are plated on an osmotically stabilized media without the use of agar overlay.

168. The method of claim 163, wherein step (5) is accomplished by overlaying an appropriate selection drug antibiotic onto the growing cells, when one of the unique genomic mutations is linked to a selectable marker which results in resistance to the selection drug.

169. The method of claim 163, wherein step (5) is accomplished by genotyping, when none of the unique genomic mutations is linked to a selectable marker.

170. The method of claim 163, wherein genetic mutations derived from more than two strains are randomly consolidated during a single consolidation process.

171. The method of claim 163, wherein in step (2) the protoplasts are initially collected by centrifuging at a speed about 5000xg for about 5 minutes.

172. The method of claim 163, wherein the method does not comprise of filtrating the protoplasts through cotton wool.

173. The method of claim 163, wherein the fused protoplasts are recovered on a R2YE media rather than top-agar.

174. The method of claim 173, wherein the R2YE media comprises 0.5M sorbitol and 0.5M mannose.

175. A method of targeted genome editing in a Saccharopolyspora strain, comprising :

a) introducing a plasmid comprising a selection marker, a counterselection marker, a DNA fragment having homology to the genomic locus of the Saccharopolyspora strain to be edited, and plasmid backbone sequence into a base Saccharopolyspora strain;

b) selecting for Saccharopolyspora strains with integration event based on the presence of the selection marker in the genome;

c) selecting for Saccharopolyspora strains having the plasmid backbone looped out based on the absence of the counterselection marker gene, wherein the counterselection marker is a sacB gene or a pheS gene.

176. The method of claim 175, wherein the resulted Saccharopolyspora strain with edited genome has better performance compared to the parent strain without the editing.

177. The method of claim 176, wherein the resulted Saccharopolyspora strain has increased spinosyn production compared to the parent strain without the editing.

178. The method of claim 175, wherein the sacB gene is codon-optimized for

Saccharopolyspora spinosa.

179. The method of claim 178, wherein the sacB gene encodes an amino acid sequence with 90% sequence identity to the amino acid sequence encoded by SEQ ID No. 146.

180. The method of claim 175, wherein the pheS gene is codon-optimized for

Saccharopolyspora spinosa.

181. The method of claim 180, wherein the pheS gene encodes an amino acid sequence with 90% sequence identity to the amino acid encoded by SEQ ID No. 147 or SEQ ID No. 148.

182. A method of transferring genetic material from donor microorganism cells to recipient cells of a Saccharopolyspora microorganism, wherein the method comprises the steps of:

1) Optionally, subculturing recipient cells to late- exponential or stationary phase;

2) Optionally, subculturing donor cells to mid-exponential phase;

3) Combining donor and recipient cells;

4) Plating donor and recipient cell mixture on conjugation media;

5) Incubating plates to allow cells to conjugate;

6) Applying antibiotic selection against donor cells;

7) Applyig antibiotic selection against non-integrated recipient cells; and

8) further incubating plates to allow for the outgrowth of integrated recipient cells.

183. The method of claim 182, wherein the donor microorganism cells are E. coli cells.

184. The method of claim 182, wherein at least two, three, four, five, six, seven or more of the following conditions are utilized:

1) recipient cells are washed before conjugating;

2) donor cells and recipient cells are conjugated at a temperature of about 30 °C;

3) recipient cells are sub-cultured for at least about 48 hours before conjugating;

4) the ratio of donor cells : recipient cells for conjugation is about 1 : 0.6 to 1 : 1.0;

5) an antibiotic drug for selection against the donor cells is delivered to the mixture about 15 to 24 hours after the donor cells and the recipient cells are mixed;

6) an antibiotic drug for selection against the recipient cells is delivered to the mixture about 40 to 48 hours after the donor cells and the recipient cells are mixed;

7) the conjugation media plated with donor and recipient cell mixture is dried for at least about 3 hours to 10 hours;

8) the conjugation media comprises at least about 3 g/L glucose;

9) the concentration of donor cells is about OD600= 0.1 to 0.6;

10) the concentration of recipient cells is about OD540= 5.0 to 15.0;

185. The method of claim 184, wherein the antibiotic drug for selection against the donor cells is nalidixic, and the concentration is about 50 to about 150 μg/ml.

186. The method of claim 185, wherein the antibiotic drug for selection against the donor cells is nalidixic, and the concentration is about 100 μg/ml.

187. The method of claim 184, wherein the antibiotic drug for selection against the recipient cells is apramycin, and the concentration is about 50 to about 250 μg/ml.

188. The method of claim 187, wherein the antibiotic drug for selection against the recipient cells is apramycin, and the concentration is about 100 μg/ml.

189. The method of claim 182, wherein the method is performed in a high-throughput process.

190. The method of claim 189, wherein the method is performed on a 48-well Q-trays.

191. The method of claim 189, wherein the high-throughput process is automated.

192. The method of claim 191 , where the mixture of donor cells and recipient cells is a liquid mixture, and ample volume of the liquid mixture is plated on the medium with a rocking motion, wherein the liquid mixture is dispersed over the whole area of the medium.

193. The method of claim 191, wherein the method comprises automated process of transferring exconjugants by colony picking with yeast pins for subsequent inoculation of recipient cells with integrated DNA provided by the donor cells.

194. The method of claim 193, the colony picking is performed in either a dipping motion, or a stirring motion.

195. The method of claim 184, wherein the conjugating media is a modified ISP4 media comprising about 3-10 g/L glucose.

196. The method of claim 184, wherein the total number of donor cells or recipient cells in the mixture is about 5 x 106 to about 9 X l O6

197. The method of claim 182, wherein the method is performed with at least four of the following conditions:

1) recipient cells are washed before conjugating;

2) donor cells and recipient cells are conjugated at a temperature of about 30 °C;

3) recipient cells are sub-cultured for at least about 48 hours before conjugating;

4) the ratio of donor cells : recipient cells for conjugation is about 1 :0.8;

5) an antibiotic drug for selection against the donor cells is delivered to the mixture about 20 hours after the donor cells and the recipient cells are mixed;

6) the amount of the donor cells or the amount of the recipient cells in the mixture is about 7 x 106, and

7) the conjugation media comprises about 6 g/L glucose.

198. A method of targeted genomic editing in a Saccharopolyspora strain, resulting in a scarless Saccharopolyspora strain containing a genetic variation at a targeted genomic locus, comprising: a) introducing a plasmid into a Saccharopolyspora strain, said plasmid comprising:

i. a selection marker,

ii. a counterselection marker ,

iii. a DNA fragment containing a genetic variation to be integrated into the Saccharopolyspora genome at a target locus, said DNA fragment having homology arms to the target genomic locus flanking the desired genetic variation, and iv. plasmid backbone sequence;

b) selecting for a Saccharopolyspora strain that has undergone an initial homologous recombination and has the genetic variation integrated into the target locus based on the presence of the selection marker in the genome; and

c) selecting for a Saccharopolyspora strain that has the genetic variation integrated into the target locus, but has undergone an additional homologous recombination that loops-out the plasmid backbone, based on the absence of the counterselection marker,

wherein said targeted genomic locus may comprise any region of the Saccharopolyspora genome, including genomic regions that do not contain repeating segments of encoding DNA modules.

199. The method of claim 198, wherein the plasmid does not comprise a temperature sensitive replicon.

200. The method of claim 198, wherein the plasmid does not comprise an origin of replication.

201. The method of claim 198, wherein the selection step (c) is performed without replication of the integrated plasmid.

202. The method of claim 198, wherein the plasmid is a single homologous recombination vector.

203. The method of claim 198, wherein the plasmid is a double homologous recombination vector.

204. The method of claim 198, wherein the counterselection marker is a sacB gene or a pheS gene.

205. The method of claim 204, wherein the sacB gene or pheS gene is codon-optimized for

Saccharopolyspora spinosa.

206. The method of claim 205, wherein the sacB gene encodes an amino acid sequence with 90% identity to the amino acid sequence encoded by SEQ ID NO. 146.

207. The method of claim 205, wherein the pheS gene encodes an amino acid sequence with 90% sequence identity to the amino acid encoded by SEQ ID NO. 147 or SEQ ID NO. 148.

208. The method of claim 198, wherein the plasmid is introduced into the Saccharopolyspora strain by transformation.

209. The method of claim 198, wherein the transformation is a protoplast transformation.

210. The method of claim 198, wherein the plasmid is introduced into the Saccharopolyspora strain by conjugation, wherein the Saccharopolyspora strain is a recipient cell, and a donor cell comprising the plasmid transfers the plasmid to the Saccharopolyspora strain.

211. The method of claim 198, wherein the conjugation is based on an E. coli donor cell comprising the plasmid.

212. The method of claim 198, wherein the target locus is a locus associated with production of a compound of interest in the Saccharopolyspora strain.

213. The method of claim 198, wherein the resulting Saccharopolyspora strain has increased production of a compound of interest compared to a control strain without the genomic editing.

214. The method of claim 212 or claim 213, wherein the compound of interest is a spinosyn.

215. The method of claim 198, wherein the method is performed as a high-throughput procedure.