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 functionally agnostic method for rehabilitating and improving the phenotypic performance of a production microbial strain, which does not require prior knowledge regarding the underlying function of a given genetic variation, the method 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 the parental lineage microbial strain to add one or more of the identified genetic variations present in the production microbial strain, or perturbing the genome of the production microbial strain to remove one or more of the identified genetic variations not present in the parental lineage microbial strain, to create an initial library of microbial strains, wherein each strain in the initial library comprises a genetic variation from the plurality of identified genetic variations between the parental lineage microbial strain and the production microbial strain, and wherein at least one of the strains of the initial library comprises a genetic variation that does not have a known enzymatic or regulatory function;
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; and
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 method enables analyzing genome-wide combinatorial effects of genetic variations across multiple disparate genomic regions, including uncharacterized genes and/or genes of no known function.
2. 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.
3. The method according to claim 1 , wherein the subsequent library is a subset of a full combinatorial library of the initial library.
4. The method according to claim 1 , wherein the subsequent library is a subset of a full combinatorial library of a preceding library.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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, protein, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.
10. The method according to claim 1 , wherein said production microbial strain is a prokaryote.
11. 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, 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.
12. The method according to claim 1 , wherein said production microbial strain is Corynebacterium glutamicum.
13. The method according to claim 1 , wherein said production microbial strain is a eukaryote.
14. 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, Diplodia, 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.
15. The method according to claim 1 , wherein said production microbial strain is Aspergillus niger.