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1. (WO2018226880) PLATE-FORME D'INGÉNIERIE GÉNOMIQUE HTP PERMETTANT D'AMÉLIORER ESCHERICHIA COLI
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CLAIMS

What is claimed is:

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

a. perturbing the genomes of an initial plurality of E. coli microbes having the same genomic strain background, to thereby create an initial HTP genetic design E. coli strain library comprising individual E. coli strains with unique genetic variations; b. screening and selecting individual strains of the initial HTP genetic design E. coli strain library for the desired phenotype;

c. providing a subsequent plurality of E. coli microbes that each comprise a unique combination of genetic variation, said genetic variation selected from the genetic variation present in at least two individual E. coli strains screened in the preceding step, to thereby create a subsequent HTP genetic design E. coli strain library; d. screening and selecting individual E. coli strains of the subsequent HTP genetic design E. coli strain library for the desired phenotype; and

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

2. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design E. coli 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 protein solubility tag microbial strain library, a protein degradation tag microbial strain library and any combination thereof.

3. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design E. coli strain library comprises a promoter swap microbial strain library.

4. The HTP method of genomic engineering according to claim 1 or 2, wherein the initial HTP genetic design E. coli strain library comprises a promoter swap microbial strain library that contains at least one bicistronic design (BCD) regulatory sequence.

5. The HTP method of genomic engineering according to claim 1, wherein the initial HTP genetic design E. coli strain library comprises a SNP swap microbial strain library.

6. The HTP method of genomic engineering according to claim 1 or 2, wherein the initial HTP genetic design E. coli strain library comprises a microbial strain library that comprises:

a. at least one polynucleotide encoding for a chimeric biosynthetic enzyme, wherein said chimeric biosynthetic enzyme comprises an enzyme involved in a regulatory pathway in E. coli translationally fused to a DNA binding domain capable of binding a DNA binding site; and

b. at least one DNA scaffold sequence that comprises the DNA binding site.

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

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

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

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

11. 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, and any combination thereof.

12. The HTP method of genomic engineering according to claim 1 , wherein the initial plurality of E. coli microbes comprise unique genetic variations derived from an industrial production E. coli strain.

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

14. A method for generating a SNP swap E. coli strain library, comprising the steps of:

a. providing a reference E. coli strain and a second E. coli strain, wherein the second E. coli 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 E. coli strain; and

b. perturbing the genome of either the reference E. coli strain, or the second E. coli strain, to thereby create an initial SNP swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual 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 E. coli strain and the second E. coli strain.

15. The method for generating a SNP swap ?. coli strain library according to claim 14, wherein the genome of the reference E. coli 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 E. coli strain.

16. The method for generating a SNP swap ?. coli strain library according to claim 14, wherein the genome of the second E. coli 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 E. coli strain.

17. The method for generating a SNP swap ?. coli strain library according to any one of claims 14-16, wherein the resultant plurality of individual E. coli strains with unique genetic variations, together comprise a full combinatorial library of all the identified genetic variations between the reference E. coli strain and the second E. coli strain.

18. The method for generating a SNP swap ?. coli strain library according to any one of claims 14-16, wherein the resultant plurality of individual E. coli strains with unique genetic variations, together comprise a subset of a full combinatorial library of all the identified genetic variations between the reference E. coli strain and the second E. coli strain.

19. A method for rehabilitating and improving the phenotypic performance of a production E. coli strain, comprising the steps of:

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

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

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

e. screening and selecting individual strains of the subsequent library for 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 an E. coli strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production E. coli strain, wherein each subsequent iteration creates a new 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 E. coli strains of a preceding library.

20. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the initial library of E. coli strains is a full combinatorial library comprising all of the identified genetic variations between the parental lineage E. coli strain and the production E. coli strain.

21. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the initial library of E. coli strains is a subset of a full combinatorial library comprising a subset of the identified genetic variations between the parental lineage E. coli strain and the production E. coli strain.

22. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the subsequent library of E. coli strains is a full combinatorial library of the initial library.

23. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the subsequent library of E. coli strains is a subset of a full combinatorial library of the initial library.

24. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the subsequent library of E. coli strains is a full combinatorial library of a preceding library.

25. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the subsequent library of E. coli strains is a subset of a full combinatorial library of a preceding library.

26. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the genome of the parental lineage E. coli 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 E. coli strain.

27. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the genome of the production E. coli 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 E. coli strain.

28. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to any one of claims 19-25, 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.

29. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

30. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli 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 E. coli strain.

31. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, 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.

32. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, 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.

33. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, wherein the identified genetic variations further comprise artificial promoter swap genetic variations from a promoter swap library.

34. The method for rehabilitating and improving the phenotypic performance of a production E. coli strain according to claim 19, further comprising:

engineering the genome of at least one microbial strain of either:

the initial library of E. coli strains, or

a subsequent library of E. coli strains,

to comprise one or more promoters from a promoter ladder operably linked to an endogenous E. coli target gene.

35. A method for generating a promoter swap E. coli strain library, comprising the steps of: a. providing a plurality of target genes endogenous to a base E. coli strain, and a promoter ladder, wherein said promoter ladder comprises a plurality of promoters exhibiting different expression profiles in the base E. coli strain; and

b. engineering the genome of the base E. coli strain, to thereby create an initial promoter swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli 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 E. coli strain.

36. The method for generating a promoter swap E. coli strain library according to claim 35, wherein at least one of the plurality of promoters comprises a bicistronic design (BCD) regulatory sequence.

37. A promoter swap method for improving the phenotypic performance of a production E. coli strain, comprising the steps of:

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

b. engineering the genome of the base E. coli strain, to thereby create an initial promoter swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli 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 E. coli strain;

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

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

e. screening and selecting individual E. coli strains of the subsequent promoter swap E. coli strain library for 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 an E. coli strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production E. coli strain, wherein each subsequent iteration creates a new promoter swap E. coli 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 E. coli strains of a preceding library.

38. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, wherein the subsequent promoter swap E. coli strain library is a full combinatorial library of the initial promoter swap E. coli strain library.

39. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, wherein the subsequent promoter swap E. coli strain library is a subset of a full combinatorial library of the initial promoter swap E. coli strain library.

40. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, wherein the subsequent promoter swap E. coli strain library is a full combinatorial library of a preceding promoter swap E. coli strain library.

41. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, wherein the subsequent promoter swap E. coli strain library is a subset of a full combinatorial library of a preceding promoter swap E. coli strain library.

42. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 37-41, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent promoter swap E. coli strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

43. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 37-41, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent promoter swap E. coli strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

44. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, 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.

45. The promoter swap method for improving the phenotypic performance of a production E. coli strain according to claim 37, 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.

46. A method for generating a terminator swap E. coli strain library, comprising the steps of: a. providing a plurality of target genes endogenous to a base E. coli strain, and a terminator ladder, wherein said terminator ladder comprises a plurality of terminators exhibiting different expression profiles in the base E. coli strain; and

b. engineering the genome of the base E. coli strain, to thereby create an initial terminator swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli 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 E. coli strain.

47. A terminator swap method for improving the phenotypic performance of a production E. coli strain, comprising the steps of:

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

b. engineering the genome of the base E. coli strain, to thereby create an initial terminator swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli 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 E. coli strain;

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

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

e. screening and selecting individual E. coli strains of the subsequent terminator swap E. coli strain library for 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 an E. coli strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production E. coli strain, wherein each subsequent iteration creates a new terminator swap E. coli 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 E. coli strains of a preceding library.

48. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, wherein the subsequent terminator swap E. coli strain library is a full combinatorial library of the initial terminator swap E. coli strain library.

49. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, wherein the subsequent terminator swap E. coli strain library is a subset of a full combinatorial library of the initial terminator swap E. coli strain library.

50. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, wherein the subsequent terminator swap E. coli strain library is a full combinatorial library of a preceding terminator swap E. coli strain library.

51. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, wherein the subsequent terminator swap E. coli strain

library is a subset of a full combinatorial library of a preceding terminator swap E. coli strain library.

52. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 47-51, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent terminator swap E. coli strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

53. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 47-51, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent terminator swap E. coli strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

54. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, 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.

55. The terminator swap method for improving the phenotypic performance of a production E. coli strain according to claim 47, 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.

56. A system for colocalizing bios nthetic enzymes from a hiosynthetic pathway in an li coli host cell, said system comprising:

a. two or more chimeric enzyme proteins involved in an enzymatic reaction, each chimeric enzyme protein comprising an enzyme portion coupled to a DNA binding domain portion; and

b. a DN A scaffold comprising

i. one or more su bunks, each subunit comprising two or more different DNA binding sites separated by at least one nucleic acid spacer; wherein the chimeric enzyme proteins are recruited to the DNA scaffold by their coupled DNA binding domain portions, each of which bind at least one DNA binding site in the DNA scaffold.

57. The system of claim 56, wherem the DNA binding domain portions of the chimeric enzyme proteins comprise zinc finger DNA binding domains and the DNA binding sites of the DN scaffold comprise corresponding zinc finger binding sequences.

58. The system of claim 56, wherein the enzyme portion of each of the two or more chimeric enzyme proteins is coupled to its respective DNA binding domain portion via a polypeptide linker sequence

59. The system of claim 56, wherem the enzyme portion of each of the two or more chimeric enzyme proteins is coupled to its respective DNA binding domain portion via its ammo- terminus or i s carboxy-terminus.

60. The system of claim 56, wherein the two or more chimeric enzyme proteins comprise enzymes of an ammo acid biosynthetic pathway.

61. A bicistronic design regulatory (BCD) sequence, said BCD sequence comprising in order: a. a promoter operably linked to;

b. a first ribosomal binding site (SD1);

c. a first cistronic sequence (Cisl);

d. a second ribosome binding site (SD2);

wherein said BCD sequence is operably linked to a target gene sequence (Cis2).

62. The BCD of claim 61, wherein SD1 and SD2 each comprise a sequence of N NGGAN N.

63. The BCD of claim 61, wherein SD1 and SD2 are different.

64. The BCD of claim 61, wherein Cisl comprises a stop codon, and wherein Cis2 comprises a start codon, and wherein the Cisl stop codon and the Cis2 start codon overlap by at least 1 nucleotide.

65. The BCD of claim 61, wherein SD2 is entirely embeded within Cisl.

66. A method for expressing two target gene proteins in a host organism, said method comprisings the steps of:

a. introducing into the host organism a first polynucleotide encoding for a first target gene protein, wherein said first polynucleotide is operably linked to a first bicistronic design regulatory (BCD) sequence according to claim 61; and b. introducing into the host organism a second polynucleotide encoding for a second target gene protein, wherein said second polynucleotide is operably linked to a second BCD according to claim 61;

wherein the first and second BCDs are identicial except for their respective Cisl sequences, and wherein the target gene proteins are expressed in the host organism at a first and second expression level, respectively.

67. The method of claim 66, wherein the first expression level is within 1.5 fold of the second expression level.

68. The method of claim 66, wherein the first and second polynucleotides experience a lower level of homologous recombination in the host cell compared to a control host cell in which the first and second polynucleotides were expressed by identical BCDs.

69. A method for generating a protein solubility tag swap E. coli strain library, comprising the steps of:

a. providing a plurality of target genes endogenous to a base E. coli strain, and a solubility tag ladder, wherein said solubility tag ladder comprises a plurality of solubility tags exhibiting different solubility profiles in the base E. coli strain; and

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

70. A protein solubility tag swap method for improving the phenotypic performance of a production E. coli strain, comprising the steps of:

a. providing a plurality of target genes endogenous to a base E. coli strain, and a solubility tag ladder, wherein said solubility tag ladder comprises a plurality of solubility tags exhibiting different expression profiles in the base E. coli strain; b. engineering the genome of the base E. coli strain, to thereby create an initial solubility tag swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli strains, wherein each of said unique genetic variations comprises one or more of the solubility tags from the solubility tag ladder operably linked to one of the target genes endogenous to the base E. coli strain;

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

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

e. screening and selecting individual E. coli strains of the subsequent solubility tag swap E. coli strain library for 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 an E. coli strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production E. coli strain, wherein each subsequent iteration creates a new solubility tag swap E. coli 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 E. coli strains of a preceding library.

71. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 70, wherein the subsequent solubility tag swap E. coli strain library is a full combinatorial library of the initial solubility tag swap E. coli strain library.

72. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 70, wherein the subsequent solubility tag swap E. coli strain library is a subset of a full combinatorial library of the initial solubility tag swap E. coli strain library.

73. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 70, wherein the subsequent solubility tag swap E. coli strain library is a full combinatorial library of a preceding solubility tag swap E. coli strain library.

74. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 70, wherein the subsequent solubility tag swap E. coli strain library is a subset of a full combinatorial library of a preceding solubility tag swap E. coli strain library.

75. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 70-74, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent solubility tag swap E. coli strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

76. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 70-74, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent solubility tag swap E. coli strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

77. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 70-74, 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.

78. The solubility tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 70-74, 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.

79. A method for generating a protein degradation tag swap E. coli strain library, comprising the steps of:

a. providing a plurality of target genes endogenous to a base E. coli strain, and a degradation tag ladder, wherein said degradation tag ladder comprises a plurality of degradation tags exhibiting different solubility profiles in the base E. coli strain; and b. engineering the genome of the base E. coli strain, to thereby create an initial degradation tag swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli strains, wherein each of said unique genetic variations comprises one or more of the degradation tags from the degradation tag ladder operably linked to one of the target genes endogenous to the base E. coli strain.

A protein degradation tag swap method for improving the phenotypic performance of a production E. coli strain, comprising the steps of:

a. providing a plurality of target genes endogenous to a base E. coli strain, and a degradation tag ladder, wherein said degradation tag ladder comprises a plurality of degradation tags exhibiting different expression profiles in the base ?. coli strain; b. engineering the genome of the base E. coli strain, to thereby create an initial degradation tag swap E. coli strain library comprising a plurality of individual E. coli strains with unique genetic variations found within each strain of said plurality of individual E. coli strains, wherein each of said unique genetic variations comprises one or more of the degradation tags from the degradation tag ladder operably linked to one of the target genes endogenous to the base E. coli strain; c. screening and selecting individual E. coli strains of the initial degradation tag swap E. coli strain library for phenotypic performance improvements over a reference E. coli strain, thereby identifying unique genetic variations that confer phenotypic performance improvements;

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

e. screening and selecting individual E. coli strains of the subsequent degradation tag swap E. coli strain library for 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 an E. coli strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production E. coli strain, wherein each subsequent iteration creates a new degradation tag swap E. coli 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 E. coli strains of a preceding library.

81. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 80, wherein the subsequent degradation tag swap E. coli strain library is a full combinatorial library of the initial degradation tag swap E. coli strain library.

82. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 80, wherein the subsequent degradation tag swap E. coli strain library is a subset of a full combinatorial library of the initial degradation tag swap E. coli strain library.

83. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 80, wherein the subsequent degradation tag swap E. coli strain library is a full combinatorial library of a preceding degradation tag swap E. coli strain library.

84. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to claim 80, wherein the subsequent degradation tag swap E. coli strain library is a subset of a full combinatorial library of a preceding degradation tag swap E. coli strain library.

85. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 80-84, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent degradation tag swap E. coli strain library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

86. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 80-84, wherein steps d)-e) are repeated until the phenotypic performance of an E. coli strain of a subsequent degradation tag swap E. coli strain library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production E. coli strain.

87. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 80-84, 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.

88. The degradation tag swap method for improving the phenotypic performance of a production E. coli strain according to any one of claims 80-84, 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.

89. A chimeric synthetic promoter operably linked to a heterologous gene for expression in a microbial host cell, wherein the chimeric synthetic promoter is 60-90 nucleotides in length and consists of a distal portion of lambda phage pR promoter, variable -35 and -10 regions of lambda phage pL and pR promoters that are each six nucleotides in length, core portions of lambda phage PL and PR promoters and a 5' UTR/Ribosomal Binding Site (RBS) portion of lambda phage pR promoter.

90. The chimeric synthetic promoter of claim 89, wherein nucleic acid sequences of the distal portion of the lambda phage pu promoter, the variable -35 and -10 regions of the lambda phage pL and pR promoters, the core portions of the the lambda phage PL and pR promoters and the 5' UTR/Ribosomal Binding Site (RBS) portion of the lambda phage pR promoter are selected from the nucleic acid sequences found in Table 1.5.

91. A chimeric synthetic promoter operably linked to a heterologous gene for expression in a microbial host cell, wherein the chimeric synthetic promoter is 60-90 nucleotides in length and consists of a distal portion of lambda phage pR promoter, variable -35 and -10 regions of lambda phage p aLnd pR promoters that are each six nucleotides in length, core portions of lambda phage PL and PR promoters and a 5' UTR/Ribosomal Binding Site (RBS) portion of the promoter of the E. coli acs gene.

92. The chimeric synthetic promoter of claim 91, wherein nucleic acid sequences of the distal portion of the lambda phage pR promoter, the variable -35 and -10 regions of the lambda phage PL and pR promoters, the core portions of the the lambda phage PL and pR promoters and the 5' UTR/Ribosomal Binding Site (RBS) portion of the promoter of the E. coli acs gene are selected from the nucleic acid sequences found in Table 1.5.

93. The chimeric synthetic promoter of any of claims 89-90, wherein the chimeric synthetic promoter consists of a nucleic acid sequence selected from SEQ ID NOs. 132-152, 159- 160, 162, 165, 174-175, 188, 190, 199-201 or 207.

94. The chimeric synthetic promoter of any of claims 91-92, wherein the chimeric synthetic promoter consists of a nucleic acid sequence selected from SEQ ID NOs. 153-158, 161, 163-164, 166-173, 176-187, 189, 191-198 or 202-206.

95. The chimeric synthetic promoter of any of claims 89-92, wherein the microbial host cell is E. coli.

96. The chimeric synthetic promoter of claim 95, wherein the heterologous gene encodes a protein product of interest found in Table 2.

97. The chimeric synthetic promoter of claim 95, wherein the heterologous gene is a gene that is part of a lysine biosynthetic pathway.

98. The chimeric synthetic promoter of claim 97, wherein the heterologous gene is selected from the asd gene, the ask gene, the horn gene, the dapA gene, the dapB gene, the dapD gene, the ddh gene, the argD gene, the dapE gene, the dapF gene, the lysA gene, the lysE gene, the zwf gene, the pgi gene, the ktk gene, the f p gene, the ppc gene, the pck gene, the ddx gene, the pyc gene or the icd gene.

99. The chimeric synthetic promoter of claim 95, wherein the heterologous gene is a gene that is part of a lycopene biosynthetic pathway.

100. The chimeric synthetic promoter of claim 99, wherein the heterologous gene is selected from the dxs gene, the ispC gene, the ispE gene, the ispD gene, the ispF gene, the ispG gene, the ispH gene, the idi gene, the ispA gene, the ispB gene, the crtE gene, the crtB gene, the crtl gene, the crtY gene , the ymgA gene, the dxr gene, the elbA gene, the gdhA gene, the appY gene, the elbB gene, or the ymgB gene.

101. The chimeric synthetic promoter of claim 95, wherein the heterologous gene encodes a biopharmaceutical or is a gene in a pathway for generating a biopharmaceutical.

102. The chimeric synthetic promoter of claim 99, wherein the biopharmaceutical is selected from humulin (rh insulin), intronA (interferon alpha2b), roferon (interferon alpha2a), humatrope (somatropin rh growth hormone), neupogen (filgrastim), detaferon (interferon beta- lb), lispro (fast-acting insulin), rapilysin (reteplase), infergen (interferon alfacon-1), glucagon, beromun (tasonermin), ontak (denileukin diftitox), lantus (long- acting insulin glargine), kineret (anakinra), natrecor (nesiritide), somavert (pegvisomant), calcitonin (recombinant calcitonin salmon), lucentis (ranibizumab), preotact (human parathyroid hormone), kyrstexxal (rh urate oxidase, PEGlyated), nivestim (filgrastim, rhGCSF), voraxaze (glucarpidase), or preos (parathyroid hormone).

103. A heterologous gene operably linked to a chimeric synthetic promoter with a nucleic acid sequence selected from SEQ ID NOs. 132-207.

104. The heterologous gene of claim 103, wherein the heterologous gene encodes a protein product of interest found in Table 2.

105. The heterologous gene of claim 103, wherein the heterologous gene is a gene that is part of a lysine biosynthetic pathway.

106. The heterologous gene of claim 105, wherein the heterologous gene is selected from the asd gene, the ask gene, the horn gene, the dapA gene, the dapB gene, the dapD gene,

the ddh gene, the argD gene, the dapE gene, the dapF gene, the lysA gene, the lysE gene, the zwf gene, the pgi gene, the ktk gene, the fbp gene, the ppc gene, the pck gene, the ddx gene, the pyc gene or the icd gene.

107. The heterologous gene of claim 103, wherein the heterologous gene is a gene that is part of a lycopene biosynthetic pathway.

108. The heterologous gene of claim 107, wherein the heterologous gene is selected from the dxs gene, the ispC gene, the ispE gene, the ispD gene, the ispF gene, the ispG gene, the ispH gene, the idi gene, the ispA gene, the ispB gene, the crtE gene, the crtB gene, the crtl gene, the crtY gene , the ymgA gene, the dxr gene, the elbA gene, the gdhA gene, the appY gene, the elbB gene, or the ymgB gene.

109. The heterologous gene of claim 103, wherein the heterologous gene encodes a biopharmaceutical or is a gene in a pathway for generating a biopharmaceutical.

110. The heterologous gene of claim 109, wherein the biopharmaceutical is selected from humulin (rh insulin), intronA (interferon alpha2b), roferon (interferon alpha2a), humatrope (somatropin rh growth hormone), neupogen (filgrastim), detaferon (interferon beta- lb), lispro (fast-acting insulin), rapilysin (reteplase), infergen (interferon alfacon-1), glucagon, beromun (tasonermin), ontak (denileukin diftitox), lantus (long-acting insulin glargine), kineret (anakinra), natrecor (nesiritide), somavert (pegvisomant), calcitonin (recombinant calcitonin salmon), lucentis (ranibizumab), preotact (human parathyroid hormone), kyrstexxal (rh urate oxidase, PEGlyated), nivestim (filgrastim, rhGCSF), voraxaze (glucarpidase), or preos (parathyroid hormone).