Microbial strain improvement by a HTP genomic engineering platform
The present disclosure provides a HTP microbial genomic engineering platform that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. The HTP genomic engineering platform described herein is microbial strain host agnostic and therefore can be implemented across taxa. Furthermore, the disclosed platform can be implemented to modulate or improve any microbial host parameter of interest.
1. A method for rehabilitating and improving the phenotypic performance of a production microbial strain, comprising the steps of:
a. providing a parental lineage microbial strain and a production microbial strain derived therefrom, wherein the production microbial strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, not present in the parental lineage microbial strain;
b. perturbing the genome of either the parental lineage microbial strain, or the production microbial strain, to create an initial library of microbial strains, wherein each strain in the initial library comprises a genetic variation that is unique from amongst the plurality of identified genetic variations between the parental lineage microbial strain and the production microbial strain;
c. screening and selecting individual strains of the initial library for phenotypic performance improvements over a reference microbial strain, thereby identifying genetic variations that confer phenotypic performance improvements;
d. providing a subsequent plurality of microbes that each comprise a combination of genetic variations from the genetic variations present in at least two individual microbial strains screened in the preceding step, to thereby create a subsequent library of microbial strains;
e. screening and selecting individual strains of the subsequent library for phenotypic performance improvements over the reference microbial strain, thereby identifying combinations of genetic variation that confer additional phenotypic performance improvements;
f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a microbial strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production microbial 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 microbial strains of a preceding library; and
wherein the genome of at least one microbial strain of either: the initial library or a subsequent library, comprises one or more promoters from a promoter ladder operably linked to an endogenous target gene.
2. The method according to claim 1 , wherein the initial library is a full combinatorial library comprising all of the identified genetic variations between the parental lineage microbial strain and the production microbial strain.
3. The method according to claim 1 , wherein the initial library is a subset of a full combinatorial library comprising a subset of the identified genetic variations between the parental lineage microbial strain and the production microbial strain.
4. The method according to claim 1 , wherein the subsequent library is a full combinatorial library of the initial library.
5. The method according to claim 1 , wherein the subsequent library is a subset of a full combinatorial library of the initial library.
6. The method according to claim 1 , wherein the subsequent library is a full combinatorial library of a preceding library.
7. The method according to claim 1 , wherein the subsequent library is a subset of a full combinatorial library of a preceding library.
8. The method according to claim 1 , wherein the genome of the parental lineage microbial 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 microbial strain.
9. The method according to claim 1 , wherein the genome of the production microbial 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 microbial strain.
10. The method 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 combinations thereof.
11. The method according to claim 1 , wherein steps d)-e) are repeated until the phenotypic performance of a microbial strain of a subsequent library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production microbial strain.
12. The method according to claim 1 , wherein steps d)-e) are repeated until the phenotypic performance of a microbial 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 microbial strain.
13. The method according to claim 1 , 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.
14. The method according to claim 1 , 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.
15. The method according to claim 1 , wherein said production microbial strain is a prokaryote.
16. The method according to claim 1 , wherein said production microbial strain is from a genus selected from the group consisting of: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia , and Zymomonas.
17. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum.
18. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum , and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine.
19. The method according to claim 1 , wherein said production microbial strain is a eukaryote.
20. The method according to claim 1 , wherein said production microbial strain is from a genus selected from the group consisting of: Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium , and Volvariella.
21. The method according to claim 1 , wherein said production microbial strain is Aspergillus niger.
22. The method according to claim 1 , wherein said production microbial strain is Aspergillus niger , and wherein the improved phenotypic performance of step f) is increased or more efficient production of citric acid.
23. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum
and the one or more promoters are selected from the group of promoters of SEQ ID NOs: 1-8.
24. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum , and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine
and the one or more promoters are selected from the group of promoters of SEQ ID NOs: 1-8.
25. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum , and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine
and the endogenous target gene is selected from the group consisting of: PTS, zwf, pgi, tkt, fbp, ppc, pyc, aspB, ask, asd, dapA, dapB, dapD, cg0931, dapE, dapF, ddh, lysA, and lysE, and the one or more promoters are selected from the group of promoters of SEQ ID NOs: 1-8.
26. A system for rehabilitating and improving the phenotypic performance of a production microbial strain, the system comprising:
one or more processors; and
one or more memories operatively coupled to at least one of the one or more processors and having instructions stored thereon that, when executed by at least one of the one or more processors, cause the system to:
a. provide a parental lineage microbial strain and a production microbial strain derived therefrom, wherein the production microbial strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, not present in the parental lineage microbial strain;
b. perturb the genome of either the parental lineage microbial strain, or the production microbial strain, to create an initial library of microbial strains, wherein each strain in the initial library comprises a genetic variation that is unique from amongst the plurality of identified genetic variations between the parental lineage microbial strain and the production microbial strain;
c. screen and select individual strains of the initial library for phenotypic performance improvements over a reference microbial strain, thereby identifying genetic variations that confer phenotypic performance improvements;
d. provide a subsequent plurality of microbes that each comprise a combination of genetic variations from the genetic variations present in at least two individual microbial strains screened in the preceding step, to thereby create a subsequent library of microbial strains;
e. screen and select individual strains of the subsequent library for phenotypic performance improvements over the reference microbial strain, thereby identifying combinations of genetic variation that confer additional phenotypic performance improvements; and
f. repeat steps d)-e) one or more times, in a linear or non-linear fashion, until a microbial strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production microbial 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 microbial strains of a preceding library.