Compositions and methods to deliver iron-sulfur cluster to native and non-native enzymes

Abstract

The present invention provides engineered organisms that can deliver iron-sulfur clusters to enzymes expressed in the same organism. The enzymes can be native or non-native to the organism. The engineered organisms comprises one or more non-native iron-sulfur cluster producing systems. Also provided herein are engineered organisms that can express various heterologous polypeptides or deliver iron-sulfur clusters to the heterologous polypeptides.

Description

WSGR Docket No. 66551-702.601COMPOSITIONS AND METHODS TO DELIVER IRON-SULFUR CLUSTER TO NATIVE AND NON-NATIVE ENZYMESCROSS-REFERENCE

[0001] This application claims the benefit of Untied States Patent Application No. 63 / 702,725, filed October 03, 2024, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] This disclosure is related to expressing heterologous polypeptides in engineered organisms.SUMMARY

[0003] Provided herein, are engineered organisms. In an aspect, an engineered organism comprises: a nitrogenase; and one or more non-native iron-sulfur cluster producing systems; wherein the nitrogenase is expressed in a species of the engineered organism.

[0004] In some embodiments, the species is klebsiella and / or paenibacillus . In some embodiments, the nitrogenase is a klebsiella nitrogenase or a paenibacillus nitrogenase. In some embodiments, the engineered organism is / ■ / coli. In some embodiments, the engineered organism is yeast. In some embodiments, the yeast is S. cerevisiae. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise an eukaryotic iron-sulfur cluster producing system. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SUF from E. coli, SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, a full SUF system in E. coli, or a full SUF system in Monocercomonoides exilis, or a combination thereof. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufCB fusion only from Blastocystis and one or more polypeptides from SUF from E. coli. In some embodiments, the engineered organism is an eukaryote of a different species from the Blastocystis. In some embodiments, iron-sulfur clusters produced by the one or more non- native iron-sulfur cluster producing systems are configured-metalate at least a portion of the nitrogenase.

[0005] Provided herein, are engineered organisms. In an aspect, an engineered organism comprises: a non-native prokaryotic enzyme; and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic enzyme is expressed in a species of the engineered organism.WSGR Docket No. 66551-702.601

[0006] In some embodiments, the engineered organism is yeast. In some embodiments, the yeast is S. cerevisiae. In some embodiments, the engineered organism is a plant. In some embodiments, the non-native prokaryotic enzyme is an iron-sulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster. In some embodiments, one of the one or more non-native iron-sulfur cluster producing systems comprises a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof. In some embodiments, the one or more non- native iron-sulfur cluster producing systems comprises SUF from E. coif SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in E. coli, a full SUF system in E. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufA. In some embodiments, the one or more non-native ironsulfur cluster producing systems comprise SufA from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufB. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufB from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufC. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufC from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufS. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufS from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufE. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufE from E. coli. In some embodiments, none of the one or more non-native iron-sulfur cluster producing systems comprises SufD from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in A. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis. In some embodiments, the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism. In some embodiments, the engineered organism is a second eukaryotic organism different from the eukaryotic organism. In some embodiments, the one or more non-WSGR Docket No. 66551-702.601 native iron-sulfur cluster producing systems are configured-produce more iron-sulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine. In some embodiments, iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a portion of the non-native prokaryotic enzyme.

[0007] Provided herein, are engineered organisms. In an aspect, an engineered organism comprises: a non-native prokaryotic system of Entner-Doudoroff pathway (EDP); and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic system is expressed in a species of the engineered organism.

[0008] In some embodiments, the engineered organism is yeast. In some embodiments, the yeast is S. cerevisiae. In some embodiments, the non-native prokaryotic system comprises an ironsulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster. In some embodiments, one of the one or more non-native iron-sulfur cluster producing systems comprises a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SUF from E. coli, SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in E. coli, a full SUF system in E. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufA. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufA from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufB. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufB from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufC. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise SufC from E. coli. In some embodiments, the one or more non- native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufS. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufS from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises SufE. In some embodiments, he one or more non-nativeWSGR Docket No. 66551-702.601 iron-sulfur cluster producing systems comprises SufE from E. coli. In some embodiments, none of the one or more non-native iron-sulfur cluster producing systems comprises SufD from E. coli. In some embodiments, the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in E. coli. In some embodiments, the one or more non-native ironsulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis. In some embodiments, the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism. In some embodiments, the engineered organism is a second eukaryotic organism different from the eukaryotic organism. In some embodiments, the one or more non- native iron-sulfur cluster producing systems are configured-produce more iron-sulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine. In some embodiments, iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a prokaryotic enzyme of the prokaryotic system.

[0009] Provided herein, are compositions. In an aspect, a composition comprises (a) any engineered organisms as described herein, (b) iron; and (c) cysteine.

[0010] In one aspect, provided herein is an engineered organism comprising: a nitrogenase; and one or more non-native iron-sulfur cluster producing systems; wherein the nitrogenase is expressed in a species of the engineered organism.

[0011] In another aspect, provided herein is an engineered organism comprising: a non-native prokaryotic enzyme; and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic enzyme is expressed in a species of the engineered organism.

[0012] In still another aspect, provided herein is an engineered organism comprising: a non- native prokaryotic system of Entner-Doudoroff pathway (EDP); and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic system is expressed in a species of the engineered organism.

[0013] In some embodiments, the engineered organism or the target organism disclosed herein is an agricultural organism.

[0014] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictiveWSGR Docket No. 66551-702.601INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0017] FIG. 1 shows an example Entner-Doudoroff (ED) pathway in an engineered organism.

[0018] FIG. 2 shows examples of activities of nitrogenase in E.coli in the presence or absence of various iron-sulfur cluster producing systems.

[0019] FIG. 3 shows additional examples of activities of nitrogenase in E.coli in the presence or absence of various iron-sulfur cluster producing systems.

[0020] FIG. 4 shows 6-phosphogluconate dehydratase (EDD) activities in yeast in the presence or absence of iron-sulfur producing systems.

[0021] FIG. 5 shows 6-phosphogluconate dehydratase (EDD) activities in yeast in the presence or absence of iron or sulfur.

[0022] FIG. 6 shows additional examples of 6-phosphogluconate dehydratase (EDD) activities in yeast in the presence or absence of various iron-sulfur producing systems.

[0023] FIG. 7 shows examples of 6-phosphogluconate dehydratase (EDD) activities in different yeast in the presence or absence of various iron-sulfur producing systems.

[0024] FIG. 8 shows 6-phosphogluconate dehydratase (EDD) activities in yeast in the presence or absence of TCR tags.

[0025] FIG. 9 shows 6-phosphogluconate dehydratase (EDD) activities in yeast in the presence or absence of additional iron-sulfur producing systems.

[0026] FIG. 10 shows nitrogenase activities of example polypeptides when grown with various iron-sulfur cluster biosynthesis systems.

[0027] FIG. 11 shows nitrogenase activities of example polypeptides expressed in yeast alongside various example FeS systems.WSGR Docket No. 66551-702.601

[0028] FIG. 12 shows nitrogenase activities of example polypeptides expressed in yeast alongside various example genomically-integrated FeS systems.

[0029] FIG. 13 shows nitrogenase activities of example polypeptides expressed in and purified from different example yeast strains.

[0030] FIG. 14 shows the EDD activities of example polypeptides purified from different yeast strains expressing EDD alongside various example FeS systems.

[0031] FIG. 15 shows the radical S-adenosyl-L-methionine (SAM) enzyme activities of example polypeptides purified from yeast strains expressing an example FeS system.DETAILED DESCRIPTIONOverview

[0032] Cloning and modern synthetic biology tools can facilitate heterologous expression of different polypeptides across genera, phyla, or kingdoms that can have diverse therapeutic, cosmetic, or industrial applications. One such type of polypeptides can comprise enzymes or those of the entire enzymatic pathways(a pathway when referring to enzymes, or an enzymatic pathway, can comprise the collection of enzymes, polypeptides, or components that can form series of linked biochemical or enzymatic reactions). Even with these new advancements, delivering the cofactor for these enzymes has been problematic. One such cofactor, iron-sulfur clusters, are extremely difficult to be incorporated when proteins are expressed heterologously. This is an especially difficult problem when expressing prokaryotic iron-sulfur cluster enzymes in eukaryotes. From an efficiency or economic perspective, this problem has prevented the expression of high-value pathways in eukaryotes.

[0033] As used herein the term “heterologous” when used in reference to a polynucleotide, a gene, or a polypeptide refers to a polynucleotide, gene, or polypeptide not originated in the host organism. “Heterologous” can also refer to a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. The heterologous polynucleotide or gene may be introduced into the host organism by, e.g., gene transfer. A heterologous gene may include a native coding region with non-native regulatory regions that is reintroduced into the native host. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0034] Additionally, continuous biomanufacturing systems or biomanufacturing using fermentation can face many stability challenges. For example, over time, biomass and other solids can accumulate, lowering run-times and resulting in inconsistent quality issues

[0035] Additionally, cell stress can cause genetic drift, altering the genetic makeup of the population and overall productivity. Stressing a biological system can create selection pressure,WSGR Docket No. 66551-702.601 potentially leading to genetic drift. While microbes are robust and capable of efficient production, high output levels and specific product choices can contribute to this pressure. Ensuring genetic stability can maintain consistent productivity and quality.

[0036] Furthermore, microbial contamination introduced through feedlines can also disrupt production, leading to inefficiencies and downtime. Contamination from other microbes can be a significant problem with continuous biomanufacturing systems, and can cause problems such as loss of productivity, extended downtime. . . etc.

[0037] These issues are hurdles in the campaign to achieve consistent, high-yield production that can facilitate biomanufacturing platforms to better compete with petrochemicals commercially, incentivizing development of crucial biomanufacturing infrastructure. Overcoming these issues can greatly improve the value of heterologous expression of polypeptides.

[0038] One way to address these issues can comprise engineering an organism’s pathways to reduce biomass and energy use, thereby extending the duration of the production run while still achieving high product yields. Such engineering can comprise engineering and streamlining the organism’s metabolism by redirecting cell resources into production instead of biomass. The engineering can extend run-times by decreasing unneeded biomass accumulation, reduce genetic drift rate by slowing specific growth rate, or lower contamination risk by decreasing fermenter dilution rates.

[0039] In one aspect, the present disclosure provides engineered organisms that comprise ironsulfur delivery pathway. In some embodiments, Iron-sulfur (Fe-S or FeS) clusters can be cofactors or prosthetic groups to facilitate the activity of the class of proteins or polypeptides that comprise them (which is also referred to as iron-sulfur cluster proteins). In the class of ironsulfur cluster containing proteins, the iron-sulfur clusters have been found to play several roles: When proteins of this class are first synthesized by the cell, they lack the iron-sulfur clusters required for their proper function. These form of the proteins are referred to as apoproteins. Ironsulfur clusters are made in a series of reactions by proteins involved in iron-sulfur cluster biosynthesis and are transferred to the apoproteins to form the functional iron-sulfur cluster containing holoproteins.

[0040] As used herein here Fe-S refers to a iron-sulfur. xFe-xS or [xFe-xS] refers to x number of iron(s) bound to x number of sulfur(s).

[0041] The source of sulfur in an iron-sulfur cluster comes from cysteine and is accomplished by the action of cysteine desulfurases. One type desulfurase in the iron-sulfur cluster (ISC) system is called IscS, a pyridoxal phosphate-dependent enzyme that proposed an enzyme-bound persulfide functional group as a source of sulfur that can be incorporated into iron-sulfurWSGR Docket No. 66551-702.601 clusters. Similar desulfurases exist in the nitrogen fixation and sulfur assimilation or formation systems.

[0042] After these iron-sulfur clusters are synthesized on a scaffold protein and transferred to a recipient protein (apoprotein); the apoprotein becomes a functional iron-sulfur protein. The cluster formation step occurs in the cytoplasm of prokaryotic organisms or in the mitochondria of eukaryotic organisms. In yeast and other higher organisms the iron-sulfur clusters are transported out of the mitochondrion before being incorporated into the recipient proteins, such as, extramitochondrial enzymes. These higher organisms also possess a set of proteins involved in the iron-sulfur clusters transport and incorporation processes that are not homologous to proteins found in prokaryotic systems. The present disclosure comprise engineered organisms co-expressing non-native proteins in need of iron-sulfur cluster assembly and non-native ironsulfur cluster formation systems in the yeast cytosol, then inserts the iron-sulfur clusters into apo-iron-sulfur proteins in cytosol, thereby providing assembly of active iron-sulfur proteins in the yeast cytosol. As used herein, the term “non-native” when referring to a component in an organism refers to the component that is not originated in the organism. In some embodiments, a non-native polypeptide within an organism is polypeptide that is not originated in the genome of that organism. As used herein, the term “native” when referring to a component in an organism refers to the component that is originated in the organism.

[0043] Examples of iron-sulfur proteins include, but are not limited to, ferredoxins, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, hydrogenases, coenzyme Q - cytochrome c reductase, succinate - coenzyme Q reductase, nitrogenase, or other heterologous polypeptides as described herein.

[0044] The canonical iron-sulfur delivery system in yeast and animals is found in the mitochondria. In plants iron-sulfur cluster systems are in the mitochondria and chloroplasts, These system are known to only deliver iron-sulfur cluster specifically to their own proteins. Even though several researchers have attempted to solve this problem, utilizing various strategies, none have been successful. As shown in the Examples, the compositions and methods disclosed herein provide a solution to this problem and can achieve higher activity for a prokaryotic iron-sulfur cluster containing protein expressed in yeast when compared with the native yeast. Additional steps to help deliver the iron-sulfur cluster to the desired iron-sulfur cluster-utilizing enzyme in yeast or other eukaryotes can be helpful to further improve the biomanufacturing process in yeast or in other processes as outlined before. The biogenesis of iron sulfur clusters has been studied in the bacteria A. coli and A. vinelandii and yeast S. cerevisiae.WSGR Docket No. 66551-702.601

[0045] Facilitating iron-sulfur cluster delivery to prokaryotic polypeptide or pathways expressed in eukaryotes can have transformative benefits, such as but not limited to (1) introducing a new pathway that previously had not been shown to work efficiently in eukaryotes (such as nitrogen fixation) or (2) replacing inefficient eukaryotic pathways with more efficient ones for generating heterologous polypeptides or molecules.

[0046] In one aspect, the present disclosure provides engineered organisms that comprise an iron-sulfur (Fe-S) protein and an engineered system to deliver iron-sulfur (Fe-S) clusters to the Fe-S protein.

[0047] In one aspect, the present disclosure provides engineered organisms that comprise the Entner-Doudoroff (ED) pathway into yeast metabolism. This ED pathway can produce less ATP per glucose molecule than traditional glycolysis. The ED pathway can provide an alternative to glycolysis (i.e., the Embden-Meyerhof-Parnas (EMP) pathway) to extract energy from glucose. The ED pathway may be found in prokaryotes. The ED pathway can be more desirable than glycolysis for certain biomanufacturing purposes.

[0048] In some embodiments, the ED pathway can convert 6-phosphogluconate (6-PG) to glyceraldehyde-3 -phosphate (GAP) and pyruvate in two steps catalyzed by two enzymes: a 6- PG dehydratase (EDD) and a 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EDA), respectively. The ED pathway may be restricted to prokaryotes. In some embodiments, the EDD genes may be absent in eukaryotes. Under conditions of nitrogen limitation, the ED pathway may be favored by single-cell prokaryotes when compared with the glycolysis (EMP pathway). Completing the ED pathway in yeast can comprise expressing one or more enzymes, EDD (6- phosphogluconate dehydratase) and EDA (KDPG aldolase).

[0049] Integrating the ED pathway in yeast can limit resources directed to biomass, thereby resulting in greater production efficiency of a particular polypeptide. The engineered organisms can express one or more enzymes of the ED pathway and an engineered system to deliver ironsulfur (Fe-S) clusters to the enzymes, and overcoming a challenge of prior attempts to express a functional ED pathway in yeast. The ED pathway can replace glycolysis in eukaryotes, where the efficiency gain can come from not having organisms such as yeast utilize an ATP towards biomass. By fine-tuning the flux through the ED pathway, the methods can maintain the desired rate of production while extending production time. Extended production at lower growth rates may reduce genetic drift and contamination risks. While yeast can more genetically stable, introducing the ED pathway can make them even more genetically stable, thereby enhancing the genetic stability of the corresponding biomanufacturing systems. Additionally, the method can comprise engineering organism to better compete with other organisms or gain the capability to grow on a carbon source that is less likely to be utilized by other microbes, thereby improvingWSGR Docket No. 66551-702.601 resistance to contamination. For example, incorporation of the ED pathway into yeasts may facilitate the yeasts to grow on gluconate, a less commonly used carbon source, as a means to further reduce contamination.

[0050] Introducing the ED pathway into eukaryote or yeast have been unsuccessful due to the difficulty of delivering an iron-sulfur (Fe-S) cluster as a cofactor to the EDD enzyme.

[0051] In one aspect, the present disclosure provides engineered organisms that comprise the nitrogenase.

[0052] In one aspect, the present disclosure provides engineered organisms that comprise the methylerythritol phosphate pathway (MEP). The MEP pathway can replace the native the mevalonate pathway.

[0053] In one aspect, the present disclosure provides engineered organisms that comprise the Radical SAM enzyme(s). The radical SAM enzyme(s) can facilitate chemistry that does not work efficiently or at all currently in eukaryotes. The radical SAM enzymes or pathway can facilitate antibiotic production, herbicides for plants, or nutritional compounds such as Biotin.

[0054] In one aspect, the present disclosure provides engineered organisms that comprise the Fe-Hydrogenases. The engineered organisms can be used for biohydrogen gas production.

[0055] In one aspect, the present disclosure provides engineered organisms that comprise the Rieske dioxygenases. The engineered organisms can be used bioremediation of aromatic pollutants.

[0056] In one aspect, the present disclosure provides engineered organisms that comprise the actonitase-family dehydratases and isomerases. The engineered organisms can be used for specialty chemical synthesis such as bio-based polymers.

[0057] In one aspect, the present disclosure provides engineered organisms that comprise the 3- dehydroxyshikimate dehydratase. The engineered organisms can be used for creating agricultural feedstocks by reducing or removing lignin from a lignocellulose material.Engineered organisms

[0058] Provided herein, are engineered cells or engineered organisms. In some embodiments, the engineered cell or engineered organism can comprise a genetic modification. An engineered organism can comprise the engineered cell(s) cultured in vivo. An engineered organism can also comprise the engineered cell(s) cultured in vitro.

[0059] In some embodiments, the engineered cell or engineered organism can comprise greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200 or more genetic modifications. In some embodiments, the engineered cell or engineered organism can comprise less than or equal to: 1,WSGR Docket No. 66551-702.6012, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 200 genetic modifications. The genetic modifications may be made in the genome of the engineered cell or engineered organism. The genome of the engineered cell or engineered organism can comprise a chromosomal genome, a nuclear genome, a mitochondrial genome, or a chloroplastic genome. The genetic modifications may be integrated into the genome of the engineered cell or engineered organism. The genetic modifications may not be integrated into the genome of the engineered cell or engineered organism.

[0060] In some embodiments, the genetic modification can comprise a nucleic acid encoding a heterologous polypeptide. In some embodiments, the engineered cell or engineered organism can comprise a heterologous polypeptide. In some embodiments, the engineered cell or engineered organism can comprise greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more different heterologous polypeptides. In some embodiments, the engineered cell or engineered organism can comprise less than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 more different heterologous polypeptides. In some embodiments, the engineered cell or engineered organism may be configured to express a heterologous polypeptide. In some embodiments, the engineered cell or engineered organism may be configured to express greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more different heterologous polypeptides. In some embodiments, the engineered cell or engineered organism may be configured to express at less than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 more different heterologous polypeptides.

[0061] In some embodiments, the protein level of the heterologous polypeptide within the engineered cell or engineered organism may be greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold higher than that of a control cell or organism. The control cell or organism may be the same species or same cell type of the engineered cell or engineered organism but does not have the engineering or genetic modification of engineeredWSGR Docket No. 66551-702.601 cell or engineered organism. In some embodiments, the control cell or organism may not have any genetic modification or engineering. In some embodiments, the control cell or organism may be a wildtype or natural counterpart of the engineered cell or engineered organism. In some embodiments, for engineered organisms comprising any iron-sulfur protein as described herein, the control organism may be the same species or same cell type of the engineered cell or engineered organism but does not have the iron-sulfur biosynthesis pathway. In some embodiments, the protein level of the heterologous polypeptide within the engineered cell or engineered organism may be less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000- fold, or 1000000-fold higher than that of the control cell or organism.

[0062] In some embodiments, the engineered cell or engineered organism may have greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more copies of the nucleic acid sequences encoding the heterologous polypeptides. In some embodiments, the engineered cell or engineered organism may have less than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies of the nucleic acid sequences encoding the heterologous polypeptides.

[0063] In some embodiments, the heterologous polypeptide can comprise a polypeptide or a polypeptide sequence originated in the host cell or host organism, wherein the host cell or host organism is the organism that is being engineered. For example, the heterologous polypeptide can comprise a polypeptide or a polypeptide sequence of an endogenous protein or polypeptide of the host cell or host organism (or form the same species of the host cell or host organism). In some embodiments, the heterologous polypeptide can comprise a polypeptide or a polypeptide sequence not originated in the host cell or host organism. For example, the heterologous polypeptide can comprise a polypeptide or a polypeptide sequence of an endogenous protein or polypeptide of an organism that is a different species of the host cell or host organism. In some cases, the engineered organism may comprise more than or equal to two heterologous polypeptides, and the two heterologous polypeptides may originate from two different organism that is different from the host organism. The two organisms may comprise two different prokaryotes. The two organisms may comprise two different eukaryotes. The two organisms may comprise a eukaryote and a prokaryote.WSGR Docket No. 66551-702.601

[0064] In some embodiments, the heterologous polypeptide may be expressed using a heterologous expression module. In some embodiments, the heterologous expression module is integrated in the genome of the cell. In some embodiment, the heterologous expression module is not integrated in the genome of the cell. In some embodiment, the heterologous expression module heterologous transcriptional unit is carried on a vector or plasmid. In some embodiment, the heterologous expression module is expressed using an inducible transcription promoter. In some embodiment, the heterologous expression module is expressed using a constitutive transcription promoter. In some embodiment, the heterologous expression module comprises various non-coding sequence elements. The non-coding sequence elements can comprise introns, terminators, 3’UTR, or translational regulatory elements for expressing the heterologous polypeptides.

[0065] An engineered organism can comprise a prokaryote. An engineered cell or engineered organism can comprise an eukaryote. An engineered organism can comprise an archaea, bacteria, yeast, plant, or animal. An engineered organism can comprise an archaea. An engineered organism can comprise a bacteria. An engineered organism can comprise a yeast. An engineered organism can comprise plant. An engineered organism can comprise an animal. An engineered organism can comprise a bacterial, yeast, or plant species comprising or expressing the heterologous polypeptide. The yeast can comprise Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces, or Pichia cells. The yeast can comprise A cerevisiae. The bacteria can comprise d, coli. In some embodiments, the engineered cell or organism can comprise yeast. In some embodiments, the engineered cell or organism can comprise an agricultural cell or organism (e.g., corn, wheat, rice, soybean, cotton, or grass). The engineered cell or organism can comprise corn. The engineered cell or organism can comprise wheat. The engineered cell or organism can comprise rice. The engineered cell or organism can comprise soybean. The engineered cell or organism can comprise cotton. The engineered cell or organism can comprise grass.

[0066] When the engineered cell or organism is a prokaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from a different species of the engineered cell or organism. When the engineered cell or organism is a prokaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from a different prokaryotic species of the engineered cell or organism. When the engineered cell or organism is an eukaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from a different species of the engineered cell or organism. When the engineered cell or organism is an eukaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from a different eukaryotic species of the engineered cell or organism.WSGR Docket No. 66551-702.601When the engineered cell or organism is a prokaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from an eukaryote. When the engineered cell or organism is an eukaryote, the engineered cell or organism can comprise a heterologous polypeptide originated from an prokaryote.Iron-sulfur cluster

[0067] In some embodiments, the engineered organism or cell can comprise a polypeptide of the iron-sulfur (Fe-S) cluster biosynthesis pathway. As used herein the term “iron-sulfur cluster producing system,” “iron-sulfur cluster biosynthesis pathway,” or “iron-sulfur cluster biosynthesis” refers to biosynthesis of Fe-S clusters. The pathway can comprise the assembly or loading of Fe-S clusters. The “Fe-S biosynthesis genes” or “Fe-S cluster biosynthesis proteins” can comprise the polynucleotides or genes; or the encoded polypeptides that facilitate the biosynthesis of the Fe-S clusters. The “Fe-S cluster biosynthesis genes” or “Fe-S cluster biosynthesis proteins” can comprise the polynucleotides or genes; or the encoded polypeptides that facilitate the assembly and loading of Fe-S clusters. In some embodiments, the heterologous polypeptide as described herein can comprise one or more polypeptides of the Fe-S cluster biosynthesis pathway.

[0068] In some embodiments, the engineered organism or cell can comprise a Fe-S protein or polypeptide. As used herein the term “Fe-S cluster protein or polypeptide” or “Fe-S cluster protein or polypeptide” refers to a protein or polypeptide that binds an Fe-S cluster, wherein the binding of the Fe-S cluster can facilitate its activity. In some embodiments, the heterologous polypeptide as described herein can comprise one or more Fe-S polypeptides.

[0069] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can facilitate or increase the activity of a heterologous polypeptide as described herein. In some embodiments, the activity of the heterologous polypeptide can depend on the particular polypeptide, such as any of those described herein, using the method that ca measure the activity of the heterologous polypeptide, such as any methods as disclosed herein. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the activity of a heterologous polypeptide by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000- fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the one or more polypeptides of the Fe-S cluster biosynthesis pathway. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the activity of a heterologous polypeptide by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %,WSGR Docket No. 66551-702.601150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the one or more polypeptides of the Fe-S cluster biosynthesis pathway.

[0070] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can facilitate or increase the stability of a heterologous polypeptide as described herein. A stability of a polypeptide can be assayed via the level of the polypeptide present (1) within an organism or cell or (2) in vitro. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the stability of a heterologous polypeptide by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the one or more polypeptides of the Fe-S cluster biosynthesis pathway. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the stability of a heterologous polypeptide by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the one or more polypeptides of the Fe-S cluster biosynthesis pathway.

[0071] In some embodiments, a Fe-S cluster can facilitate or increase the activity of a heterologous polypeptide as described herein. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the activity of a heterologous polypeptide by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the Fe-S cluster. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the activity of a heterologous polypeptide by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the Fe-S cluster.

[0072] In some embodiments, a Fe-S cluster can facilitate or increase the stability of a heterologous polypeptide as described herein. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the stability of a heterologous polypeptide by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %,WSGR Docket No. 66551-702.60160 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the Fe-S cluster. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the stability of a heterologous polypeptide by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the heterologous polypeptide without the Fe-S cluster.

[0073] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can facilitate or increase the activity of an enzyme as described herein as described herein. In some embodiments, the enzyme catalyzes or facilitates the enzyme reaction of substrate — > product, and the activity of any enzyme described herein can be assayed by measuring (1) the consumption of the substrate; (2) the formation of the product; (3) a combination of ( 1 )-(2). In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the activity of an enzyme as described herein by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the one or more polypeptides of the Fe-S cluster biosynthesis pathway. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the activity of an enzyme as described herein by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the one or more polypeptides of the Fe-S cluster biosynthesis pathway.

[0074] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can facilitate or increase the stability of an enzyme as described herein as described herein. A stability of a polypeptide can be assayed via the level of the polypeptide present (1) within an organism or cell or (2) in vitro. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the stability of an enzyme as described herein by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000- fold, relative to that of the enzyme as described herein without the one or more polypeptides ofWSGR Docket No. 66551-702.601 the Fe-S cluster biosynthesis pathway. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can increase the stability of an enzyme as described herein by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the one or more polypeptides of the Fe-S cluster biosynthesis pathway.

[0075] In some embodiments, a Fe-S cluster can facilitate or increase the activity of an enzyme as described herein as described herein. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the activity of an enzyme as described herein by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the Fe-S cluster. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the activity of an enzyme as described herein by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the Fe-S cluster.

[0076] In some embodiments, a Fe-S cluster can facilitate or increase the stability of an enzyme as described herein as described herein. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the stability of an enzyme as described herein by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the Fe-S cluster. In some embodiments, the Fe-S cluster biosynthesis pathway can increase the stability of an enzyme as described herein by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enzyme as described herein without the Fe-S cluster.

[0077] In some embodiments, an engineered organism may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more iron-sulfur cluster,WSGR Docket No. 66551-702.601 relative to that or a control organism. In some embodiments, an engineered organism may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more iron-sulfur cluster, relative to that or a control organism.

[0078] In some embodiments, an engineered organism, when supplemented with iron and cysteine, may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more iron-sulfur cluster, relative to that the engineered organism when without supplemented with iron and cysteine. In some embodiments, an engineered organism, when supplemented with iron and cysteine, may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more iron-sulfur cluster, relative to that the engineered organism when without supplemented with iron and cysteine.

[0079] In some embodiments, an engineered organism comprising the iron-sulfur cluster biosynthesis and a heterologous polypeptide, may facilitate the metalation of the heterologous polypeptide by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more iron-sulfur cluster, relative to that or a control organism comprising the heterologous polypeptide but does not comprise the iron-sulfur cluster biosynthesis. In some embodiments, an engineered organism comprising the iron-sulfur cluster biosynthesis and a heterologous polypeptide, may facilitate the metalation of the heterologous polypeptide by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more ironsulfur cluster, relative to that or a control organism comprising the heterologous polypeptide but does not comprise the iron-sulfur cluster biosynthesis.

[0080] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different polypeptides. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise less than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different polypeptides. In someWSGR Docket No. 66551-702.601 embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different polypeptides from any Fe-S cluster biosynthesis pathway as described herein. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise less than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different polypeptides from any Fe-S cluster biosynthesis pathway as described herein. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway of the engineered organism can comprise the complete set of the Fe-S cluster biosynthesis pathway. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway of the engineered organism may not comprise the complete set of the Fe-S cluster biosynthesis pathway.

[0081] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: nitrogen fixation (NIF) system; iron-sulfur cluster (ISC) system; sulfur formation (SUF or suf) system; or cytosolic iron-sulfur assembly (CIA). In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the NIF system. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the ISC system. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the SUF system. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the CIA system. The NIF system can comprise the prokaryotic NIF system. The NIF system can comprise the bacterial NIF system. The ISC system can comprise the prokaryotic ISC system. The ISC system can comprise the eukaryotic ISC system. The ISC system can comprise the bacterial ISC system. The ISC system can comprise the mitochondrial ISC system. The SUF system can comprise the eukaryotic SUF system. The SUF system can comprise the plant SUF system. The SUF system can comprise the plastid SUF system. The SUF system can comprise the bacterial SUF system. The CIA system can comprise the eukaryotic CIA system. The CIA system can comprise the animal CIA system. The CIA system can comprise the yeast CIA system. The CIA system can comprise the plant QUA system. The CIA system can comprise the cytosolic CIA system. The CIA system can comprise the nuclear CIA system.

[0082] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: the SUF system or an iron-sulfur cluster carrier protein (mrp); Suf; SufCB fusion; or NifUS. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the SUF system or an iron-sulfur cluster carrier protein (mrp). In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SUF In some embodiments, the one or more polypeptides ofWSGR Docket No. 66551-702.601 the Fe-S cluster biosynthesis pathway can comprise SufCB fusion. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifUS.

[0083] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: SUF from E. coli,' SufCB fusion from Blastocystis NifUS from Entamoeba histolytica, NifUS from Klebsiella, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, a SUF system in E. coli, or a SUF system in Monocercomonoides exilis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: the SUF system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough,' SUF from E. coli,' SufCB fusion from Blastocystis,' NifUS from Entamoeba histolytica, NifUS from Klebsiella, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, a full SUF system in E. coli, or a full SUF system in Monocercomonoides exilis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the SUF system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SUF from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB fusion from Blastocystis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifUS from Entamoeba histolytica. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifUS from Klebsiella. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a SUF system in A. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a SUF system in Monocercomonoides exilis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a full SUF system in A. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a full SUF system in Monocercomonoides exilis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the SUF system from Desulfovibrio vulgaris Hildenborough. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the mrp from Desulfovibrio vulgaris Hildenborough.

[0084] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: SufCB or SufASE. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB. In someWSGR Docket No. 66551-702.601 embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufASE. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB and SufASE. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway may not comprise SufD. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB and SufASE but may not comprise SufD. SufCB, SufASE or SufD can comprise those from a prokaryote. SufCB, SufASE or SufD can comprise those from a bacteria.

[0085] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: SufCB from Blastocystis or SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB from Blastocystis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB from Blastocystis and SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway may not comprise SufD from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB from Blastocystis and SufASE from E. coli but may not comprise SufD from E. coli.

[0086] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: SufCB fusion from Blastocystis or SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB fusion from Blastocystis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB fusion from Blastocystis and SufASE from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway may not comprise SufD from E. coli. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise SufCB fusion from Blastocystis and SufASE from E. coli but may not comprise SufD from E. coli.

[0087] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise one or more of: complete or partial SUF systems from archaea; complete or partial SUF systems from eukaryotic plastid; complete or partial SUF systems from eukaryotic cytosolic SUF systems; complete or partial SUF systems from amitochondriate eukaryotes; alternative chaperones; Fe-S carriers from plants; Fe-S carriers from yeast; fusions of core SUF genes; nifU from nif strains; Fe-S carriers and accessory proteins from eukaryoticWSGR Docket No. 66551-702.601 and prokaryotic ISC system; or alternative desulfurases from prokaryotic or eukaryotic SUF, nifU or ISC systems.

[0088] In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise complete or partial SUF systems from archaea. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise complete or partial SUF systems from eukaryotic plastid. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise complete or partial SUF systems from eukaryotic cytosolic SUF systems. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise complete or partial SUF systems from amitochondriate eukaryotes; alternative chaperones. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise Fe-S carriers from plants; Fe-S carriers from yeast. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise fusions of core SUF genes; nifU from nif strains. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise Fe-S carriers and accessory proteins from eukaryotic and prokaryotic ISC system. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise alternative desulfurases from prokaryotic or eukaryotic SUF, nifU or ISC systems. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide of niF. In some cases, a polypeptide of NiF or a polypeptide encode by a gene of nif can comprise multiple [4Fe-4S] for cofactor biosynthesis. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifB, NifH, NifK, NifD, NifE, NifN, NifS, or NifU. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifH. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifK. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifD. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifE. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifN. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NiFB. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NiFU. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NiFS. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise NifB, NifH, NifK, NifD, NifE, NifN, NifS, and NifU. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by nifB, nifH, nifK,WSGR Docket No. 66551-702.601 nifD, niffi, nifN, nifS, or nifU. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by niffi. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by nifH. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by niffi. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by nifD. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by niffi. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by nifN. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by nifS. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise a polypeptide encoded by niffi. In some embodiments, the one or more polypeptides of the Fe-S cluster biosynthesis pathway can comprise the polypeptides encoded by niffi, nifH, niffi, nifD, niffi, nifN, nifS, and niffi.

[0089] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the Fe-S cluster biosynthesis pathway as described herein. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the Fe-S cluster biosynthesis pathway as described herein. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide of the Fe-S cluster biosynthesis pathway as described herein.

[0090] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufCB, SufCB fusion, from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufCB, SufCB fusion, from E. coli. In some embodiments, the engineeredWSGR Docket No. 66551-702.601 organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide of SufCB, SufCB fusion, from E. coli.

[0091] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufABCDSE. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufABCDSE. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufABCDSE.

[0092] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufASE. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufASE. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufASE.

[0093] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufA. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufA. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufA.

[0094] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufB. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95WSGR Docket No. 66551-702.601%, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufB. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufB.

[0095] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufC. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufC. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufC.

[0096] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufD. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufD. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufD.

[0097] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufS. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufS. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufS.

[0098] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufE. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40WSGR Docket No. 66551-702.601%, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufE. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufE.

[0099] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from Blastocystis . In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from Blastocystis. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from Blastocystis.

[0100] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide of SufCB, SufCB fusion, or any of those from E. coli.

[0101] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufABCDSE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufABCDSE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufABCDSE or any of those from E. coli.WSGR Docket No. 66551-702.601

[0102] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufASE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufASE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufASE or any of those from E. coli.

[0103] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufA or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufA or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufA or any of those from E. coli.

[0104] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufB or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufB or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufB or any of those from E. coli.

[0105] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufC or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than orWSGR Docket No. 66551-702.601 equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufC or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufC or any of those from E. coli.

[0106] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufD or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufD or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufD or any of those from E. coli.

[0107] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufS or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufS or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufS or any of those from E. coli.

[0108] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of SufE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of SufE or any of those from E. coli. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide SufE or any of those from E. coli.WSGR Docket No. 66551-702.601

[0109] In some embodiments, the Fe-S biosynthesis pathway can comprise a polypeptide or a polypeptide sequence originated in the host cell or host organism, wherein the host cell or host organism is the organism that is being engineered. For example, the heterologous polypeptide of the Fe-S biosynthesis pathway can comprise a polypeptide or a polypeptide sequence of an endogenous protein or polypeptide of the host cell or host organism (or form the same species of the host cell or host organism). In some embodiments, the heterologous polypeptide of the Fe-S biosynthesis pathway can comprise a polypeptide or a polypeptide sequence not originated in the host cell or host organism. For example, the heterologous polypeptide can comprise a polypeptide or a polypeptide sequence of an endogenous protein or polypeptide of an organism that is a different species of the host cell or host organism. In some cases, the engineered organism may comprise more than or equal to two heterologous polypeptides of the Fe-S biosynthesis pathway, and the two heterologous polypeptides may originate from two different organisms that are different from the host organism. The two organisms may comprise two different prokaryotes. The two organisms may comprise two different eukaryotes. The two organisms may comprise a eukaryote and a prokaryote. For example, as described herein, the engineered organism may comprise the SufCB from Blastocystis and the SufACSE from E. coli. Fe-S proteinEntner-Doudoroff (ED) pathway

[0110] In some embodiments, a heterologous polypeptide can comprise an polypeptide of the ED pathway. In some embodiments, the engineered organism or cell can comprise one or more polypeptides of the ED pathway. In some embodiments, the one or more polypeptides can have enzyme activities of the ED pathway.[OHl] In some embodiments, the engineered organism or cell can comprise one or more polypeptides having EDD or EDA activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having EDD activity. In some embodiments, the engineered organism or cell can comprise polypeptide having EDA activity. In some embodiments, the engineered organism or cell can comprise one or more polypeptides having EDD and EDA activities.

[0112] The EDD activity can be measured by KDPG formation. As described in Krevet et al. (Front. Bioeng. BiotechnoL 2020 Mar 20:8: 18; which is herein incorporated by reference in its entirety), KDPG formation can be assayed in a discontinuous assay in which 6-PG was incubated with EDD, and the resultant product can b assayed for NADH oxidation. For example, KDPG formation can be assayed in a discontinuous assay (0.5 ml) containing 200 mM HEPES buffer pH 8, 5 mM MnC12, 2.5 mM 6-PG and 8.1 pg purified EDD at 37°C. 100 pl samples can be removed in regular intervals, and the reaction can be stopped by addition of 10 pl 20% (w / v)WSGR Docket No. 66551-702.601TCA and incubation on ice for 10 min. After a 15 min centrifugation at 21,000 x g and 4°C, 50 pl of the supernatant can be mixed with 2 pl 2 M NaOH for neutralization. For KDPG determination 11.4 pl of these samples can be added to 100 mM HEPES buffer pH 8, containing 0.2 mM NADH, 3 U L-lactate dehydrogenase (rabbit muscle, Sigma-Aldrich) and 50 pg KDPG EDA from S. acidocaldarius (500 pl final volume) and preincubated at 37°C for 2 min. The oxidation of NADH can be followed spectrophotometrically [Analytik Jena, s(NADH) = 6.22 mM-1 cm-1]. When the reaction runs to completion 5 pl of the purified EDD (corresponding to 0.22 U and 3.6 pg of protein) can be added to determine the residual 6-PG. (see, for example, Gardner and Fridovich 1991 and Gu and Imlay 2011, each of which is herein incorporated by reference in its entirety).

[0113] In some cases, EDD activity can be measured via a two-step assay as described (in above references) via the formation of pyruvate. In the first step, EDD converts 6PG to KDPG in a 0.3mL anaerobic reaction incubated at 37C for 5-10 min and consisting of 50mM Tris, 80mM 6PG, lOOmM MgC12, and 100-200ug of cell lysate expressing EDD. Alternatively, the same reaction can be performed with 10-25ug purified EDD protein. This reaction is then halted by adding 500mL of Tris and boiling for 1 min, which converts KDPG to pyruvate. In the second step of the assay, the sample is centrifuged at 14000xg for 3 min, and pyruvate in the supernatant can be quantified in a ImL reaction consisting of 20-100uL of supemant, 1.37U lactate dehydrogenase (rabbit muscle, Sigma-Aldrich), 0.2mM NADH, and 50mM Tris. The pyruvate-dependent oxidation of NADH by LDH can be followed spectrophotometrically [6.22 mM-1 cm-1],

[0114] KDPG EDA activity can be determined by the standard coupled assay with L-lactic dehydrogenase and NADH. As described in Fong et al. (Front. Bioeng. BiotechnoL 2020 Mar 20:8: 18; which is herein incorporated by reference in its entirety), a KDPG EDA assay can comprise addition of an appropriate amount of KDG or KDPG in 50 mM potassium phosphate, pH 7.5 / 5 mM P-mercaptoethanol, to a mixture of L-lactic dehydrogenase (10 U; (EC 1.1.1.27, type II from rabbit muscle)) / NADH (0.43 mM) / aldolase (0.88 pM) in 50 mM potassium phosphate, pH 7.5 / 5 mM P-mercaptoethanol. The total reaction volume can be 800 pl. Prior to the addition of the substrate, the mixture can be preincubated at 25°C for 5 min. UV absorbance at 340 nm can be recorded continuously for 2 min and the slope of the absorbance curve during the first 30 s can be used for rate determination.

[0115] In some embodiments, the engineered organism or cell can comprise one or more of EDD or EDA. In some embodiments, the engineered organism or cell can comprise EDD. In some embodiments, the engineered organism or cell can comprise EDA. In some embodiments, the engineered organism or cell can comprise EDD and EDA. In some embodiments, theWSGR Docket No. 66551-702.601 engineered organism or cell can comprise one or more of E. coli EDD or E. coli EDA. In some embodiments, the engineered organism or cell can comprise E. coli EDD. In some embodiments, the engineered organism or cell can comprise E. coli EDA. In some embodiments, the engineered organism or cell can comprise E. coli EDD and E. coli EDA.

[0116] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the ED pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the ED pathway by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the ED pathway without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the ED pathway by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000- fold, relative to that of the ED pathway without the Fe-S cluster or Fe-S biosynthesis pathway.

[0117] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the EDD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the EDD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the EDD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDD without the Fe-S cluster or Fe-S biosynthesis pathway.

[0118] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the EDA. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the EDA by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDA without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-SWSGR Docket No. 66551-702.601 biosynthesis pathway can facilitate or increase the activity of the EDA by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDA without the Fe-S cluster or Fe-S biosynthesis pathway.

[0119] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDD without the Fe-S cluster or Fe-S biosynthesis pathway.

[0120] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDA. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDA by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDA without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the EDA by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the EDA without the Fe-S cluster or Fe-S biosynthesis pathway.

[0121] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide that has EDD activity. Percent (%) sequence identity or homology with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the referenceWSGR Docket No. 66551-702.601 polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0122] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0123] In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide that has EDD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide that has EDD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %,WSGR Docket No. 66551-702.60165 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide that has EDA activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide that has EDA activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide that has EDA activity.

[0124] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of a plant. The plant comprising a polypeptide of the ED pathway may comprise Genlisea aurea, Sesamum indicum, Erythranthe gutata, Beta vulgaris, Nelumbo nucifera, Amborella trichopoda, Phoenix dactylifera, Oryza brachyantha, Oryza Sativa Indica, Oryza Sativa Japonica, Setaria Italica, Sorghum bicolor, Zea mays, Brachypodium distachyon, Hordeum vulgare, Triticum Urartu, Aegilops tauschii, Solanum lycopersicum, Solanum tuberosum, Nicotiana sylverstris, Nicotiana tomentosiformis, Eucalyptus grandis, Cicer arietinum, Medicago truncatula, Phaseolus vulgaris, Glycine max, Glycine soja, Morus notabilis, Pyrus bretschneideri, Fragaria vesca, Cucumis sativus, Cucumis melo, Theobroma cacao, Gossypium raimondii, Gossypium arboretum, Populus euphratica, Populus trichocarpa, Jatropha curcas, or Ricinus communis. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of a plant. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of a plant.

[0125] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of a bacteria. The bacteria comprising a polypeptide of the ED pathway may comprise E. coli, Sorangium cellulosum, Candidatus Solibacter usitatus, Dictyoglomus thermophilum, Planctomyces limnophilus, Opitutus terrae, Thermomicrobium roseum, Gramella forsetii, Clostridium perfringens, Lactobacillus brevis, Thermotoga petrophila, Rubrobacter xylanophilus , TreponemaWSGR Docket No. 66551-702.601 pallidum, Aminobacterium colombiense, Pseudomonas entomophila, Sphingopyxis alaskensis, Helicobacter pylori, Burkhoderia cenocepacia, Meiothermus ruber, or Candidatus Phytoplaama. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of a bacteria. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of a bacteria.

[0126] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of a cyanobacteria. The cyanobacteria comprising a polypeptide of the ED pathway may comprise Prochlorococcus marinus, Gloeobacter violaceus, Thermosynechococcus elongatus, Fibrobacter succinogenes, Synechococcus elongatus, Synechococcus sp., Synechocystis sp., Cyanothece sp., Acaryoch / oris marina, Trichodesmium erythraeum, Nostoc sp. , Anabaena variabillis, Nostoc punctiforme, or Nostoc azollae. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of a cyanobacteria. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of a cyanobacteria.

[0127] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of an Archaea. The Archaea comprising a polypeptide of the ED pathway may comprise Haloquadratum walsbyi. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of an Archaea. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of an Archaea.WSGR Docket No. 66551-702.601

[0128] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of an algae. The algae comprising a polypeptide of the ED pathway may comprise Galideria sulphuraria or Cyanidoschyzon merolae. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of an algae. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of an algae.

[0129] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the ED pathway of a moss fern. The moss fern comprising a polypeptide of the ED pathway may comprise Physcomitrella patens or Selaginella moellendorffii . In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the ED pathway of a moss fem. In some embodiments, the engineered organism or cell can comprise to a polypeptide having 100 % sequence identity to a polypeptide of the ED pathway of a moss fem.

[0130] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to E. coli EDD. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to E. coli EDD. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to E. coli EDD. In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to E. coli EDA. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65WSGR Docket No. 66551-702.601%, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to E. coli EDA. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to E. coli EDA.

[0131] In some embodiments, the engineered organism or cell can generate one adenosine triphosphate (ATP) molecule per one glucose molecule. In some embodiments, the engineered organism or cell can generate at most one ATP molecule per one glucose molecule. In some embodiments, the engineered organism or cell can generate at less than two ATP molecules per one glucose molecule. In some embodiments, the engineered organism or cell can generate one ATP molecule per one gluconate molecule. In some embodiments, the engineered organism or cell can generate at least one ATP molecule per one gluconate molecule.

[0132] In some embodiments, the engineered organism or cell can generate one nicotinamide adenine dinucleotide phosphate (NADPH) molecule per one glucose molecule. In some embodiments, the engineered organism or cell can generate at most one NADPH molecule per one glucose molecule. In some embodiments, the engineered organism or cell can generate at less than two NADPH molecules per one glucose molecule. In some embodiments, the engineered organism or cell can generate one NADPH molecule per one gluconate molecule. In some embodiments, the engineered organism or cell can generate at least one NADPH molecule per one gluconate molecule.

[0133] In some embodiments, the engineered organism or cell can be viable with a culture condition that does not comprise glucose. In some embodiments, the engineered organism or cell can be viable with a culture condition that comprises less than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006 %, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08 %, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of glucose per culture medium. In some embodiments, the engineered organism or cell can be viable with a culture condition that comprises greater than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006 %, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08 %, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of glucoseWSGR Docket No. 66551-702.601 per culture medium. In some embodiments, the engineered organism or cell can be viable with a culture condition that comprises less than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %,0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %,0.004 %, 0.005 %, 0.006 %, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05%, 0.06 %, 0.07 %, 0.08 %, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9%, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %,4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40%, or 50 %, by weight / weight, weight / volume, or volume / volume, of gluconate per culture medium. In some embodiments, the engineered organism or cell can be viable with a culture condition that comprises greater than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004%, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %,0.005 %, 0.006 %, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06%, 0.07 %, 0.08 %, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %,1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5%, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of gluconate per culture medium.

[0134] In some embodiments, the engineered organism or cell can proliferate with a culture condition that does not comprise glucose. The term “cell proliferation,” as used herein, can refer to a process by which a cell increases its numbers, mass, or a combination thereof. A cell can proliferate via cell division. A cell can proliferate via cell growth. A cell can proliferate via cell division and cell growth. In some embodiments, when an organism proliferates, it can increase the number, mass, or both number or mass by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, or 10000-fold. In some embodiments, when an organism proliferates, it can increase the number, mass, or both number or mass by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, or 10000-fold. In some embodiments, the engineered organism or cell can proliferate with a culture condition that comprises less than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006%, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08%, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5%, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, byWSGR Docket No. 66551-702.601 weight / weight, weight / volume, or volume / volume, of glucose per culture medium. In some embodiments, the engineered organism or cell can proliferate with a culture condition that comprises greater than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006%, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08%, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5%, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of glucose per culture medium. In some embodiments, the engineered organism or cell can proliferate with a culture condition that comprises less than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006%, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08%, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5%, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of gluconate per culture medium. In some embodiments, the engineered organism or cell can proliferate with a culture condition that comprises greater than or equal to about: 0.0001 %, 0.0002 %, 0.0003 %, 0.0004 %, 0.0005 %, 0.0006 %, 0.0007 %, 0.0008 %, 0.0009 %, 0.001 %, 0.002 %, 0.003 %, 0.004 %, 0.005 %, 0.006%, 0.007 %, 0.008 %, 0.009 %, 0.01 %, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08%, 0.09 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5%, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, or 50 %, by weight / weight, weight / volume, or volume / volume, of gluconate per culture medium.

[0135] In some embodiments, the engineered organism or cell can metabolize greater than or equal to about: 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2%, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 % 70%, 80 %, 90 %, 99 % or more intracellular glucoses into the ED pathway. In some embodiments, the engineered organism or cell can metabolize less than or equal to about: 0.1 %,0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %,7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 %, 90 %, 99 % or more intracellular glucoses into the ED pathway. In some embodiments, the engineeredWSGR Docket No. 66551-702.601 organism or cell can metabolize greater than or equal to about: 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 %, 90 %, 99 % or more intracellular glucoses into the glycolysis / EMP pathway. In some embodiments, the engineered organism or cell can metabolize less than or equal to about: 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, 1 %, 1.1 %, 1.2 %, 1.3 %, 1.4 %, 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2 %, 2.5 %, 3 %, 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 7.5 %, 8 %, 8.5 %, 9 %, 9.5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 %, 90 %, 99 % or more intracellular glucoses into the glycolysis / EMP pathway.

[0136] In some embodiments, the activity of the pentose phosphate pathway of the engineered organism or cell comprising can be less than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the activity of the pentose phosphate pathway of the engineered organism or cell comprising can be greater than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the activity of the glycolysis pathway of the engineered organism or cell comprising can be less than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the activity of the glycolysis pathway of the engineered organism or cell comprising can be greater than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell.

[0137] In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell proliferation rate that is less than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell proliferation rate that is greater than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell division rate that is less than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%WSGR Docket No. 66551-702.601 of that of a control organism or cell. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell division rate that is greater than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell growth rate that is less than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway may have a cell growth rate that is greater than or equal to about: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of that of a control organism or cell.

[0138] In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway can comprise the yeast as described herein. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway can comprise S. cerevisiae. In some embodiments, the engineered organism or cell comprising one or more polypeptides of the ED pathway can comprise Zymomonas mobilis. Nitrogenase

[0139] In some embodiments, a heterologous polypeptide can comprise a nitrogenase. A “nitrogenase” as described herein refers to a member of a category of enzymes that are capable of catalyzing dinitrogen fixation. Nitrogenases can catalyze a step in dinitrogen fixation wherein diatomic nitrogen (N2) is reduced to ammonia (NH3). In some embodiments, the heterologous polypeptide can catalyze greater than or equal to about: %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of nitrogen into ammonia. In some embodiments, the heterologous polypeptide can catalyze less than or equal to about: %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of nitrogen into ammonia. In some embodiments, nitrogenase activity can be measured by any methods as described herein.

[0140] Nitrogenases can be expressed by symbiotic genera of bacteria (e.g., rhizobacteria) found living on root systems of plants wherein the products of nitrogen fixation fertilize the soil and provide nutrients to the plants. Nitrogenases can comprise multiple components. For example, the Mo-nitrogenase is a two-component metalloenzyme comprising a molybdenum iron (MoFe) protein (NifDK) that performs the reduction reaction, and an iron (Fe) protein (NifH) that acts as an electron donor to the MoFe protein. NifH is a component that comprises iron-sulfur (Fe-S) clusters that serve as cofactors for the reduction reaction. Alternative nitrogenases can replaceWSGR Docket No. 66551-702.601 molybdenum in the metallic cofactor with either vanadium or iron to form the vanadium-iron (VFe; Vnf) nitrogenase and the iron-iron (FeFe; Anf) nitrogenase, respectively. Nitrogenases are also expressed by other bacterial genera such as Klebsiella or Paenibacillus. In some embodiments, nitrogenases from Klebsiella and / or Paenibacillus may be expressed in other bacterial species, yeast, or plants.

[0141] Nitrogenases can also perform reduction of other substrates. Acetylene can also be reduced by nitrogenase to generate ethylene. Thus, nitrogenase activity can be measured via an acetylene reduction assay (ARA). In this assay, nitrogen fixing bacteria or plants are exposed to acetylene gas for a period of time. At the end of the assay, the quantity of ethylene is quantified by any suitable method such as gas chromatography (GC). The higher quantity of ethylene produced, the greater the activity of nitrogenase. In some embodiments, nitrogenase activity is measured after greater than or equal to about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, subsequent to the initiation of the nitrogenase activity assay. In some embodiments, nitrogenase activity is measured after less than or equal to about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, subsequent to the initiation of the nitrogenase activity assay.

[0142] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the nitrogenase. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the nitrogenase by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the nitrogenase without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the nitrogenase by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the nitrogenase without the Fe-S cluster or Fe-S biosynthesis pathway.

[0143] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the nitrogenase. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the nitrogenase by greater than orWSGR Docket No. 66551-702.601 equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the nitrogenase without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the nitrogenase by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the nitrogenase without the Fe-S cluster or Fe-S biosynthesis pathway.

[0144] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide that has nitrogenase activity or a nitrogenase described below. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide that has nitrogenase activity or a nitrogenase described below. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide that has nitrogenase activity or a nitrogenase described below.

[0145] In some embodiments, the nitrogenase can comprise one or more of: nif; nifF; nifl; nifH; nifD; nifK; nifB; niffi; nifN; anfH; or anfK. In some embodiments, the nitrogenase can comprise nif. In some embodiments, the nitrogenase can comprise nifF. In some embodiments, the nitrogenase can comprise nifl. In some embodiments, the nitrogenase can comprise nifH. In some embodiments, the nitrogenase can comprise nifD. In some embodiments, the nitrogenase can comprise nifK. In some embodiments, the nitrogenase can comprise nifB. In some embodiments, the nitrogenase can comprise niffi. In some embodiments, the nitrogenase can comprise nifN. In some embodiments, the nitrogenase can comprise anfH. In some embodiments, the nitrogenase can comprise anfK. The nif can comprise the nif from Paenibacillus polymyxa. In some embodiments, the nitrogenase can comprise niffi and nifl. In some embodiments, the nitrogenase can comprise niffi and nifl from Klebsiella. The nifH can comprise nitrogenase Fe protein, [4Fe-4S], The nitrogenase can comprise nifD and nifK. The nitrogenase comprising nifD and nifK can comprise nitrogenase MoFe protein subunits, which can comprise P-clusters and FeMo-co (Fe-S core). The nitrogenase can comprise niffi and nifN.WSGR Docket No. 66551-702.601The nitrogenase comprising niffi and nifN can be scaffolding proteins comprising Fe-S cluster binding. The nitrogenase can comprise one or more of: anfH, anfD, or anfK. The nitrogenase can comprise anfH, anfD, and anfK. The nitrogenase can comprise an alternative nitrogenase system Fe-only nitrogenase. In some cases, the nitrogenase can comprise a V-nitrogenases. In some cases, the nitrogenase can comprise one or more of: vnfH, vnfD, or vnfK. In some cases, the nitrogenase can comprise vnfH, vnfD, and vnfK.Methylerythritol phosphate (MEP) pathway

[0146] In some embodiments, a heterologous polypeptide can comprise a polypeptide or enzyme of the MEP pathway.

[0147] Expression of the MEP pathway in eukaryotic organisms can provide significant advantages for isoprenoid compound production by supplementing or bypassing limitations inherent in the native mevalonate pathway. The MEP pathway provides an alternative biosynthetic route for generating the universal isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), thereby increasing overall metabolic flux toward downstream terpenoid products.

[0148] Heterologous expression of MEP pathway enzymes in industrially relevant yeast species, including Saccharomyces cerevisiae and Yarrowia lipolylica. can facilitate the production of plant-derived isoprenoids that are not naturally synthesized in these organisms. This approach can create hybrid metabolic systems capable of producing high-value compounds such as artemisinic acid, taxadiene, and specialized carotenoids including astaxanthin and P-carotene. The orthogonal nature of the MEP pathway relative to the endogenous mevalonate pathway can allow for independent metabolic control and reduced competition for shared cellular resources. Tying this system with ED can provide additional benefit since NADPH generated in the ED pathway can utilized by the MEP pathway.

[0149] While plants have both the mevalonate pathway and MEP pathway (chloroplasts), their production can be limited. Expressing the MEP pathway from prokaryotes can greatly enhance isoprenoid synthesis. This increased expression can improve plant nutritional content but can be used for industrial and pharmaceutical uses. This can comprise increasing terpenoid synthesis to provide protection against pests as well as facilitate the production of pharmaceutical precursors such as monoterpenes and sesquiterpenes.

[0150] Other pharmaceutical relevant applications include precursors for steroids, hormones, anti-cancer compound production (taxol precursors, other diterpenoids) and antimalarial compound synthesis such as artemisinin and its derivativesin. The food industry includes production of steviol glycosides (Stevia) and Mogrosides (Monk Fruit). Fragrances and flavorsWSGR Docket No. 66551-702.601(monoterpenes). MEP pathway can produce both traditional chemical feedstocks and food ingredients. The pathway can also be useful in making aviation fuel.

[0151] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the MEP pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of MEP pathway by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the MEP pathway without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the MEP pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the MEP pathway by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the MEP pathway without the Fe-S cluster or Fe-S biosynthesis pathway.

[0152] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of a polypeptide of the MEP pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of MEP pathway by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that the polypeptide of the MEP pathway without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of a polypeptide of the MEP pathway. In some embodiments, a Fe-S cluster or a Fe- S biosynthesis pathway can facilitate or increase the activity of the MEP pathway by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the polypeptide of the MEP pathway without the Fe-S cluster or Fe-S biosynthesis pathway.

[0153] In some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isoprenoid, relative to that of the of the control organism. InWSGR Docket No. 66551-702.601 some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isoprenoid, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more IPP, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more IPP, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more DMAPP, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the MEP pathway may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more DMAPP, relative to that of the of the control organism.

[0154] In some embodiments, the polypeptide of the MEP pathway can comprise one or more of: IspG (2-C-methyl-D-erythritol-2, 4-cyclodiphosphate reductase) or IspH (4-hydroxyl-3- methylbut-2-enyl diphosphate reductase). In some embodiments, the polypeptide of the MEP pathway can comprise IspG. In some embodiments, the polypeptide of the MEP pathway can comprise IspH. In some embodiments, the polypeptide of the MEP pathway can comprise IspG and IspH.

[0155] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to a polypeptide of the MEP pathway or having MEP pathway activity.WSGR Docket No. 66551-702.601In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to a polypeptide of the MEP pathway or having MEP pathway activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to a polypeptide of the MEP pathway or having MEP pathway activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to IspG. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to IspG. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to IspG. In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98%, 99 %, 99.9 % or more sequence identity to IspH. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95%, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to IspH. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to IspH.

[0156] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspH. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspH by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspH without the Fe- S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspH. In some embodiments, a Fe- S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the IspH by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspH without the Fe-S cluster or Fe-S biosynthesis pathway.WSGR Docket No. 66551-702.601

[0157] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspH. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspH by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspH without the Fe- S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspH. In some embodiments, a Fe- S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspH by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspH without the Fe-S cluster or Fe-S biosynthesis pathway.

[0158] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspG. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspG by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspG without the Fe- S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of IspG. In some embodiments, a Fe- S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the IspG by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspG without the Fe-S cluster or Fe-S biosynthesis pathway.

[0159] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspG. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspG by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspG without the Fe- S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspG. In some embodiments, a Fe- S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of IspG by lessWSGR Docket No. 66551-702.601 than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the IspG without the Fe-S cluster or Fe-S biosynthesis pathway.Radical SAM enzymes

[0160] In some embodiments, a heterologous polypeptide can comprise radical SAM enzymes.

[0161] Radical SAM enzymes can comprise massive superfamily of proteins that use an ironsulfur cluster and SAM to initiate a wide range of biochemical reactions. The radical SAM enzymes can generate a reactive radical. The radical SAM enzymes can catalyze or facilitate various chemical transformations, such as functionalizing non-activated or inactive C-H bonds. The radical SAM enzymes can facilitate various biological processes, comprising: biosynthesis of vitamins, cofactors, antibiotics, DNA repair, or the modification of proteins and RNA. Radical SAM enzymes can exist in eukaryotes. Radical SAM enzymes can exist in prokaryotes. Prokaryotic radical SAM enzymes may not be sufficiently metalated when expressed in eukaryotes. As used herein, “metalating” or “metalation” refers to the chemical process of adding one or more metal ions to a molecule Thus, the immense catalytic power of prokaryotic radical SAM enzymes is largely untapped for metabolic engineering in eukaryotes. Allowing sufficient metalation of the prokaryotic SAM enzymes can allow access to a massive toolkit of unique chemical reactions, paving the way for the sustainable production of complex antibiotics, biofuels, or other high-value compounds in eukaryotic cells. Certain prokaryotic radical SAM enzymes can be activated in eukaryotes, likely because they resemble enzymes that are similar in sequence or structure to eukaryotic counterparts.

[0162] A radical SAM enzyme can comprise class A radical SAM enzyme, class B radical SAM enzyme, or class C radical SAM enzyme. Radical SAM enzyme can comprise class A radical SAM enzyme. Radical SAM enzyme can comprise class B radical SAM enzyme. Radical SAM enzyme can comprise class C radical SAM enzyme.

[0163] A radical SAM enzyme can comprise one or more of: an enzyme comprising multiple [4Fe-4S] for cofactor biosynthesis, biotin synthase, lipoyl synthase, an enzyme of molybdopterin biosynthesis, an enzyme of thiamine pyrimidine biosynthesis, or an enzyme of thiamine biosynthesis. A radical SAM enzyme can comprise an enzyme comprising multiple [4Fe-4S] for cofactor biosynthesis,. A radical SAM enzyme can comprise biotin synthase. A radical SAM enzyme can comprise lipoyl synthase. A radical SAM enzyme can comprise an enzyme of molybdopterin biosynthesis. A radical SAM enzyme can comprise an enzyme of thiamine pyrimidine biosynthesis. A radical SAM enzyme can comprise an enzyme of thiamine biosynthesis.WSGR Docket No. 66551-702.601

[0164] An enzyme comprising multiple [4Fe-4S] for cofactor biosynthesis can comprise nifB. A biotin synthase can comprise bioB. A biotin synthase can comprise [2Fe-2S], A lipoyl synthase can comprise lipA. A lipoyl synthase can comprise multiple types of Fe-S. An enzyme of molybdopterin biosynthesis can comprise moaA. An enzyme of molybdopterin biosynthesis can comprise [4Fe-4S], An enzyme of thiamine pyrimidine biosynthesis can comprise thiC. An enzyme of [4Fe-4S], A thiamine pyrimidine biosynthesis can comprise [4Fe-4S],

[0165] A radical SAM enzyme can comprise one or more of: nifB, bioB, lipA, moaA, thiC, or thiH. A radical SAM enzyme can comprise nifB. A radical SAM enzyme can comprise bioB. A radical SAM enzyme can comprise lipA. A radical SAM enzyme can comprise moa. A radical SAM enzyme can comprise thiC. A radical SAM enzyme can comprise thiN.

[0166] bioB can comprise a biotin synthase. bioB can comprise [2Fe-2S], lipA can comprise a lipoyl synthase, radical SAM, multiple Fe-S.

[0167] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the radical SAM enzyme. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of radical SAM enzyme by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the radical SAM enzyme without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the radical SAM enzyme. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the radical SAM enzyme by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the radical SAM enzyme without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the radical SAM enzyme. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of radical SAM enzyme by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the radical SAM enzyme without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the radical SAM enzyme. In some embodiments, a Fe-S cluster or a Fe-SWSGR Docket No. 66551-702.601 biosynthesis pathway can facilitate or increase the stability of the radical SAM enzyme by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the radical SAM enzyme without the Fe-S cluster or Fe-S biosynthesis pathway.

[0168] In some embodiments, the engineered organism having one or more polypeptides of the radical SAM enzyme pathway may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isobutanol, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the radical SAM enzyme pathway may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isobutanol, relative to that of the of the control orgamsm.

[0169] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to radical SAM enzyme or a polypeptide having radical SAM enzyme activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to radical SAM enzyme or a polypeptide having radical SAM enzyme activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to radical SAM enzyme or a polypeptide having radical SAM enzyme activity.Dihydroxy Acid Dehydratase (DH AD) -dependent pathway

[0170] In some embodiments, a heterologous polypeptide can comprise a polypeptide or enzyme of the DHAD-dependent pathway. In some embodiments, a heterologous polypeptide can comprise DHAD.

[0171] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the DHAD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of DHAD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90WSGR Docket No. 66551-702.601%, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the DHAD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the DHAD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the DHAD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the DHAD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the DHAD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of DHAD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the DHAD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the DHAD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the DHAD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the DHAD without the Fe-S cluster or Fe-S biosynthesis pathway.

[0172] In some embodiments, the engineered organism having one or more polypeptides of the DHAD pathway may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isobutanol, relative to that of the of the control organism. In some embodiments, the engineered organism having one or more polypeptides of the DHAD pathway may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more isobutanol, relative to that of the of the control organism.

[0173] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % orWSGR Docket No. 66551-702.601 more sequence identity to DHAD or a polypeptide having DHAD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to DHAD or a polypeptide having DHAD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to DHAD or a polypeptide having DHAD activity.ER-mediated pathway

[0174] In some embodiments, a heterologous polypeptide can comprise a polypeptide or enzyme of the endoplasmic reticulum-mediated pathway. In some embodiments, a heterologous polypeptide can comprise enoate reductase.

[0175] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the enoate reductase. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of enoate reductase by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enoate reductase without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the enoate reductase. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the enoate reductase by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enoate reductase without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the enoate reductase. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of enoate reductase by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enoate reductase without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the enoate reductase by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-WSGR Docket No. 66551-702.601 fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the enoate reductase without the Fe-S cluster or Fe-S biosynthesis pathway.

[0176] In some embodiments, the engineered organism having enoate reductase may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more adipic acid, relative to that of the of the control organism. In some embodiments, the engineered organism having enoate reductase may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more adipic acid, relative to that of the of the control organism.

[0177] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to enoate reductase or a polypeptide having enoate reductase activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to enoate reductase or a polypeptide having enoate reductase activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to enoate reductase or a polypeptide having enoate reductase activity.Xylose to butanetriol pathway

[0178] In some embodiments, a heterologous polypeptide can comprise a polypeptide or enzyme of the xylose to butanetriol pathway. In some embodiments, a heterologous polypeptide can comprise xylonate dehydratase (XylD).

[0179] In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the XylD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the XylD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the XylD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the activity of the XylD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70WSGR Docket No. 66551-702.601%, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the Xy ID without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the XylD. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of XylD by greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the XylD without the Fe-S cluster or Fe-S biosynthesis pathway. In some embodiments, a Fe-S cluster or a Fe-S biosynthesis pathway can facilitate or increase the stability of the XylD by less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold, relative to that of the XylD without the Fe-S cluster or Fe-S biosynthesis pathway.

[0180] In some embodiments, the engineered organism having enoate reductase may generate greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more 1,2,4-butanetriol, relative to that of the of the control organism. In some embodiments, the engineered organism having enoate reductase may generate less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, or 1000000-fold more 1,2,4-butanetriol, relative to that of the of the control organism.

[0181] In some embodiments, the engineered organism or cell can comprise a polypeptide having greater than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 % or more sequence identity to XylD or a polypeptide having XylD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having less than or equal to about: 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 99.9 % sequence identity to XylD or a polypeptide having XylD activity. In some embodiments, the engineered organism or cell can comprise a polypeptide having 100 % sequence identity to XylD or a polypeptide having XylD activity.WSGR Docket No. 66551-702.601Additional Fe-S proteins

[0182] The heterologous polypeptide can comprise one or more of: 6-phosphogluconate dehydratase; pyruvate: ferredoxin oxidoreductase, [4Fe-4S] enzymes; 2-oxoglutarate: ferredoxin oxidoreductase, multiple [4Fe-4S]; [FeFe] hydrogenase subunits, all Fe-S cluster rich; formate hydrogenlyase complex, Fe-S electron transfer subunits; formate dehydrogenase H, contains [4Fe-4S]; nitrogenase Fe protein, [4Fe-4S]; nitrogenase MoFe protein subunits, contain P- clusters and FeMo-co (Fe-S core); radical SAM enzyme, multiple [4Fe-4S] for cofactor biosynthesis; scaffolding proteins, Fe-S cluster binding; alternative nitrogenase system (V- and Fe-only); biotin synthase, radical SAM, [2Fe-2S]; lipoyl synthase, radical SAM, multiple Fe-S; molybdopterin biosynthesis, radical SAM, [4Fe-4S]; thiamine pyrimidine biosynthesis, radical SAM, [4Fe-4S]; thiamine biosynthesis, radical SAM, [4Fe-4S]; 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, [4Fe-4S]; HMBPP reductase, [4Fe-4S]; subunits of RNF complex, contain multiple Fe-S centers; NADH dehydrogenase I (Complex I) Fe-S subunits; succinate dehydrogenase Fe-S subunits; Fe-Hydrogenases; Rieske dioxygenases; actonitase-family dehydratases and isomerases; or 3 -dehydroxy shikimate dehydratase. The heterologous polypeptide can comprise 6-phosphogluconate dehydratase. The heterologous polypeptide can comprise pyruvate: ferredoxin oxidoreductase, [4Fe-4S] enzymes. The heterologous polypeptide can comprise 2-oxoglutarate:ferredoxin oxidoreductase, multiple [4Fe-4S], The heterologous polypeptide can comprise [FeFe] hydrogenase subunits, all Fe-S cluster rich. The heterologous polypeptide can comprise formate hydrogenlyase complex with Fe-S electron transfer subunits. The heterologous polypeptide can comprise formate hydrogenlyase complex in E. coh. Fe-S electron transfer subunits. The heterologous polypeptide can comprise formate dehydrogenase H, contains [4Fe-4S], The heterologous polypeptide can comprise nitrogenase Fe protein, [4Fe- 4S], The heterologous polypeptide can comprise nitrogenase MoFe protein subunits, contain P- clusters and FeMo-co (Fe-S core). The heterologous polypeptide can comprise radical SAM enzyme, multiple [4Fe-4S] for cofactor biosynthesis. The heterologous polypeptide can comprise scaffolding proteins, Fe-S cluster binding. The heterologous polypeptide can comprise alternative nitrogenase system (V- and Fe-only). The heterologous polypeptide can comprise biotin synthase, radical SAM, [2Fe-2S], The heterologous polypeptide can comprise lipoyl synthase, radical SAM, multiple Fe-S. The heterologous polypeptide can comprise molybdopterin biosynthesis, radical SAM, [4Fe-4S], The heterologous polypeptide can comprise thiamine pyrimidine biosynthesis, radical SAM, [4Fe-4S], The heterologous polypeptide can comprise thiamine biosynthesis, radical SAM, [4Fe-4S], The heterologous polypeptide can comprise 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, [4Fe-4S], The heterologous polypeptide can comprise HMBPP reductase, [4Fe-4S], The heterologous polypeptide canWSGR Docket No. 66551-702.601 comprise subunits of RNF complex, contain multiple Fe-S centers. The heterologous polypeptide can comprise NADH dehydrogenase I (Complex I) Fe-S subunits. The heterologous polypeptide can comprise succinate dehydrogenase Fe-S subunits. The heterologous polypeptide can comprise Fe-Hydrogenases. The heterologous polypeptide can comprise Rieske dioxygenases. The heterologous polypeptide can comprise actonitase-family dehydratases and isomerases. The heterologous polypeptide can comprise 3 -dehydroxy shikimate dehydratase.

[0183] The heterologous polypeptide can comprise one or more of: 6-phosphogluconate dehydratase (Entner-Doudoroff pathway, [4Fe-4S]); pforA / pforB (or porA / porB); korA / korB; hydA / hydB / hydC; hycB; hycF; hycG (formate hydrogenlyase complex in E. coli); fdhF; nifH; nifD / nifK; nifB; nifE / nifN; anfH / anfD / anfK; bioB; lipA; moaA; thiC; thiH; ispG; ispH; rnfB / mfC; nuoB / nuoC / nuoD / nuoH / nuol / nuoN or sdhB / sdhC.

[0184] The heterologous polypeptide can comprise 6-phosphogluconate dehydratase (Entner- Doudoroff pathway, [4Fe-4S]). The heterologous polypeptide can comprise pforA / pforB (or porA / porB). The heterologous polypeptide can comprise korA / korB. The heterologous polypeptide can comprise hydA / hydB / hydC. The heterologous polypeptide can comprise hycB, hycF, hycG (formate hydrogenlyase complex in E. coli). The heterologous polypeptide can comprise fdhF. The heterologous polypeptide can comprise nifH. The heterologous polypeptide can comprise nifD / nifK. The heterologous polypeptide can comprise nifB. The heterologous polypeptide can comprise nifE / nifN. The heterologous polypeptide can comprise anfH / anfD / anfK. The heterologous polypeptide can comprise bioB. The heterologous polypeptide can comprise lipA. The heterologous polypeptide can comprise moaA. The heterologous polypeptide can comprise thiC. The heterologous polypeptide can comprise thiH. The heterologous polypeptide can comprise ispG. The heterologous polypeptide can comprise ispH. The heterologous polypeptide can comprise rnfB / mfC. The heterologous polypeptide can comprise nuoB / nuoC / nuoD / nuoH / nuol / nuoN. The heterologous polypeptide can comprise sdhB / sdhC.Additional engineering

[0185] In some embodiments, the genetic modification can comprise one or more of a loss-of- function mutation (LOF), a gain-of-function (GOF) mutation, or a neomorphic mutation. In some embodiments, the genetic modification can comprise a LOF mutation. In some embodiments, the genetic modification can comprise a GOF mutation. In some embodiments, the genetic modification can comprise a neomorphic mutation.

[0186] In some embodiments, the genetic modification can decrease the cell proliferation rate of the engineered organism. In some embodiments, the engineered cell or organism having the genetic modification can have a proliferation rate that is less than or equal to about: 1 %, 2 %, 3WSGR Docket No. 66551-702.601%, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %, 96 %, 97 %, 98 % or 99 % of that of a control cell or organism. In some embodiments, the engineered cell or organism having the genetic modification can have a proliferation rate that is greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %,96 %, 97 %, 98 % or 99 % of that of a control cell or organism. In some embodiments, the genetic modification can decrease the cell division rate of the engineered organism. In some embodiments, the engineered cell or organism having the genetic modification can have a division rate that is less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %, 96 %,97 %, 98 % or 99 % of that of a control cell or organism. In some embodiments, the engineered cell or organism having the genetic modification can have a division rate that is greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %, 96 %, 97 %, 98 % or 99 % of that of a control cell or organism. In some embodiments, the genetic modification can decrease the cell growth rate of the engineered organism. In some embodiments, the engineered cell or organism having the genetic modification can have a growth rate that is less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %, 96 %, 97 %, 98 % or 99 % of that of a control cell or organism. In some embodiments, the engineered cell or organism having the genetic modification can have a growth rate that is greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %< 95 %, 96 %, 97 %, 98 % or 99 % of that of a control cell or organism.

[0187] In some embodiments, the engineered cell or organism is configured to generate a product from a precursor as described herein. The products can comprise isoprenoid, IPP, DMAP. isobutanol, adipic acid, or 1,2,4- butanetri ol, as described herein.

[0188] In some cases, the engineered cell or organism can have a yield of greater than or equal to about: 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, or 5000 gram of products per liter or kilogram of the engineered cell or organism. In some cases, the engineered cell or organism can have a yield of less than or equal to about: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,WSGR Docket No. 66551-702.6012000, 3000, 4000, or 5000 gram of products per liter or kilogram of the engineered cell or organism. In some cases, the engineered cell or organism can have a yield of greater than or equal to about: 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, or 5000 gram of products per liter or kilogram of precursors. In some cases, the engineered cell or organism can have a yield of less than or equal to about: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, or 5000 gram of products per liter or kilogram of precursors.Nucleic acids and polypeptides

[0189] In some embodiments, the engineered organisms can comprise a nucleic acid (or nucleic acid molecule) that encodes the heterologous polypeptide as described herein, or the nucleic acids for generating the genetic modifications as described herein. In some embodiments, a nucleic acid can comprise a species / type of nucleic acid. In some embodiments, a nucleic acid can comprise a polynucleotide. In some embodiments, the nucleic acid can comprise a sequence of nucleotides or a nucleic acid sequence. In some embodiments, a nucleic acid can comprise one or more modified nucleotides. In some embodiments, a nucleic acid can comprise a canonical or non-canonical nucleotide. A canonical nucleotide can comprise adenosine with base types (A), cytosine (C), guanine (G), thymine (T), uracil (U), or variants thereof. When referring to a base type of a nucleotide or a polynucleotide, T and U may be interchangeable. When referring to a sequence of a nucleic acid, the sequence can comprise the complementary form of the sequence. The complementary of the nucleic acid sequence may be based on canonical base-pairing of the nucleotides or nucleic acids. In some embodiments, a nucleic acid may be single-stranded, double-stranded, triple stranded, or a combination thereof. In some embodiments, a nucleic acid may be single-stranded. In some embodiments, a nucleic acid may be double-stranded. In some embodiments, a nucleic acid can comprise single-stranded and double-stranded regions or portions thereof. A nucleic acid can comprise a deoxyribonucleic acid (DNA). A nucleic acid can comprise a ribonucleic acid (RNA).

[0190] In some embodiments, a nucleic acid may be encompassed or carried on a vector or plasmid. The term “vector” or “plasmid” can refer to a nucleic acid molecule capable of autonomous replication in a host cell, and which allow for cloning of nucleic acid molecules. A vector or plasmid can comprise a cosmid, a phagemid, a viral vector, a phage vector, a yeast vector, a mammalian vector, or the like. For example, a vector for exogenous geneWSGR Docket No. 66551-702.601 transformation may be a plasmid. In some embodiments, a vector or plasmid can comprise a nucleic acid sequence containing an origin of replication and other elements that facilitate replication, propagation, or maintenance of the nucleic acid sequence or the vector or plasmid in a host cell. In some embodiments, a vector or a plasmid provided herein is an expression vector. Expression vectors are capable of directing the expression of genes or nucleic acid sequence to which they are operatively linked. In some embodiments, an expression vector or plasmid is in the form of circular double stranded DNA molecules. In some embodiments, a peptide-DNA complex may be formed wherein the peptides comprise transit peptides that can deliver the complex into chloroplasts or mitochondria. A vector or plasmid may or may not be integrated into the genome of a host cell. In some embodiments, nucleic acid sequences of a plasmid are not integrated in a genome or chromosome of the host cell after introduction. For example, the plasmid can comprise elements for transient expression or stable expression of the nucleic acid sequences, e.g., genes or open reading frames harbored by the plasmid, in a host cell. In some embodiments, a vector is a transient expression vector. In some embodiments, a vector is a stably expressed vector that replicates autonomously in a host cell. In some embodiments, In some embodiments, nucleic acid sequences of a plasmid are integrated into a genome or chromosome of a host cell upon introduction into the host cell. Expression vectors that can be used in the methods as disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors. A vector can be a DNA or RNA vector. In some embodiments, a vector provide herein is an RNA vector that is capable of integrating into a host cell’s genome upon introduction into the host cell (e.g., via reverse transcription), for example, a retroviral vector or a lentiviral vector. Other forms of expression vectors known by those skilled in the art which serve the equivalent functions can also be used, for example, self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Example vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. In some embodiments, the vector may not be integrated into a cell or genome of a cell. In some embodiments, the nucleic acid or the vector may be used for a genetic modification or engineering as described herein.Methods for engineering genetic modifications

[0191] The genetic modification described herein can be generated by any genetic engineering systems as described herein. The heterologous polypeptide described herein can be generated into the engineered cell or organism by any genetic engineering systems as described herein.

[0192] In some cases, the genetic engineering systems can comprise using double-stranded nucleic acid-mediated homologous recombination. For example, the genetic engineering systemsWSGR Docket No. 66551-702.601 can comprise nucleic acid amplification products of the genetic engineering systems. The nucleic acid amplification products can comprise, for example, PCR-mediated amplification.

[0193] The process of introducing or incorporating a nucleic acid containing a gene of interest into a cell can be via transformation, transfection or transduction. Transformation is the process of uptake of foreign nucleic acid by a bacterial cell. This process is adapted for propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Transfection may also refer to the introduction of bacteriophage into bacterial cells. The term “transduction” is mostly used to describe the introduction of recombinant viral vector particles into target cells, while the term “infection” generally refers to natural infections of humans or animals with wild-type viruses.

[0194] The genetic engineering system can comprise CRISPR / cas. “CRISPR” or “CRISPR / Cas” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas,” as used herein, refers to a CRISPR-associated protein. A “CRISPR / Cas system” refers to a system derived from CRISPR and Cas which can be used to silence or modify a target gene. Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845. The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in gene editing applications in eukaryotic cells, the spacers are derived from the eukaryotic target gene sequence. RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1 : 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341 : 833-836.

[0195] The CRISPR / Cas systems can thus be used to edit a target gene (adding, replacing or deleting one or more base pairs), or introducing a premature stop which thus decreasesWSGR Docket No. 66551-702.601 expression of a target gene. The CRISPR / Cas system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a target promoter, sterically blocking RNA polymerases.

[0196] The genetic engineering system can comprise TALEN. “TALEN” refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene. TALENs are produced artificially by fusing a TAL effector (“TALE”) DNA binding domain, e.g., one or more TALEs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TALEs to a DNA-modifying domain, e.g., a FokI nuclease domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence. Zhang (2011), Nature Biotech. 29: 149-153. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech.29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501. TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence. Zhang (2011), Nature Biotech.29: 149-153.

[0197] The genetic engineering system can comprise Zinc Finger Nuclease. “ZFN” or “Zinc Finger Nuclease” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene. Like a TALEN, a ZFN comprises a DNA-modifying domain, e.g., a nuclease domain, e.g., a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0198] The genetic engineering system can comprise Meganuclease. The term “meganuclease” refers to an artificial nuclease which can be used to edit a target gene. Meganucleases are derived from a group of nucleases which recognize 15-40 base-pair cleavage sites. Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and / or DNA recognition. Strategies for engineering a meganuclease with altered DNA- binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art. E.g., Chevalier et al. (2002), Mol. Cell., 10:895-905; Epinat et al. (2003) Nucleic Acids Res 31 : 2952-62; Silva et al. (2006) J Mol Biol 361 : 744-54; Seligman et al. (2002) Nucleic Acids Res 30: 3870-9; Sussman et al. (2004) J Mol Biol 342: 31-41; Rosen et al. (2006) Nucleic Acids Res; Doyon et al. (2006) J Am Chem Soc 128: 2477-84; Chen et al. (2009) Protein Eng Des Sei 22:WSGR Docket No. 66551-702.601249-56; Amould S (2006) J Mol Biol.355: 443-58; Smith (2006) Nucleic Acids Res.363(2): 283-94.

[0199] A meganuclease can create a double-stranded break in the DNA, which can create a frame- shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the Meganuclease; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene, e.g., as described in Silva et al. (2011) Current Gene Therapy 11 : 11-27.

[0200] In some cases, the genetic engineering systems can comprise the components for generating the genetic modification as described herein. In some cases, the genetic engineering systems can comprise the genetic modification being engineered, as described herein.

[0201] In some cases, the genetic engineering systems may be delivered to the engineered yeast as one or more of a nucleic acid, protein / polypeptide, or Ribonucleoprotein particle (RNP). In some cases, the genetic engineering systems may be delivered to the engineered yeast via mating. For example, a host yeast that is being engineered not comprising the genetic modification system may be mated with another yeast that comprises the genetic modification system. The offspring of the mating may then carry the genetic modification system.

[0202] Standard recombinant DNA and molecular cloning techniques used herein are known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Berman, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F, M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1987), each of which references is incorporated herein by reference in its entirety.Example engineering

[0203] FIG. 1 shows a diagram of engineered yeast with the Entner-Doudoroff (ED) pathway. Glycolysis can produce two ATP per glucose molecule, one ATP is used for biomass accumulation. The proposed modifications to core metabolism are labeled with a “C”. The engineered ED pathway can produce one ATP per glucose molecule, and the ATP produced is directed towards product formation. Adding the ED pathway involves the expression of two enzymes, EDD and EDA, as well as a functional iron-sulfur delivery system. The yeast strain can be further tuned (crosses indicating down-regulation of expression) to increase the fluxWSGR Docket No. 66551-702.601 through the ED pathway (indicated by the weight of the arrows). Dotted lines depict additional coupling pathways that can be added to the reengineered central metabolism (shown in solid lines). Dashed black lines indicate delivery of iron-sulfur clusters. Although the ED pathway nets only one ATP per glucose molecule compared to the two ATP produced by the glycolysis pathway, it has the potential to increase the overall efficiency of biomanufacturing processes, such as ethanol production, by up to 20%. This potential efficiency gain is because the lower ATP yield of the ED pathway can result in less biomass production and nutrient consumption by the microbes, thereby directing more resources toward the desired product, such as ethanol.

[0204] Researchers have long sought to facilitate the ED pathway in yeast to harness these benefits but have not yet succeeded, facilitating the ED pathway in yeast can involve transferring two genes (Scheme 1) only one of which uses an iron-sulfur cluster, 6- phosphogluconate dehydratase (gene EDD from E. coif). In some embodiments, both genes from E. coli (EDA and EDD) are transferred into the target organism. If the desired flux is not obtained, additional enzymes from other species may be introduced into the yeast. For the greatest efficiency (i.e. most resources allocated to production rather than biomass), the flux through the ED pathway can be modulated in several ways. One way is to downregulate genes involved in glycolysis and the pentose phosphate pathway, which compete with the ED pathway. Another way is to express gluconate transporters to facilitate growth using gluconate as a carbon source, which may both further boost efficiency by eliminating flux through glycolysis and reduce potential contamination. Still another way is to place select glycolytic genes under switchable control in order to modulate flux between glycolysis and the ED pathway and tuning expression levels of EDD / EDA for a desired growth rates.

[0205] The choice of using yeast as the host and switching glycolysis to the ED pathway should reduce genetic drift and provide higher efficiency, which should translate to sustained production rates. The ED pathway can use both glucose and gluconate to grow: switching over to gluconate can also mitigate microbial contamination since a smaller number of microbes can grow on gluconate. Genetic drift can be measured by periodically sampling and doing metagenomic sequencing on the collected samples to observe the mutation rate in the sample. The coupling pathway can be selected such that it provides lower genetic drift. Microbial contamination can be measured by plating and growing yeast on marker-specific plates, where one or more biomarkers are introduced in yeast for detection purposes. Overall, continued modification of the biomanufacturing system can achieve sustained production rates, reduce the impact of genetic drift, and mitigate microbial contamination.

[0206] In addition to the ED pathway, the introduction of a iron-sulfur cluster delivery mechanism presents opportunities to produce other valuable metabolites in yeast. Iron-sulfurWSGR Docket No. 66551-702.601 clusters facilitate the functions of numerous enzymes, including those involved in the synthesis of amino acids, vitamins, and cofactors (Table 1). Many prior attempts at expressing these enzymes in yeast failed due to the difficulty of properly metalating heterologous enzymes in the cytosol5. With the optimization of the iron-sulfur cluster delivery in the context of the ED pathway, additional genes can be introduced to complete several other valuable pathways of prokaryotic origin: the methyl -erythritol phosphate (MEP) pathway to synthesize isoprenoids, DHAD-dependent pathway to produce isobutanol, the ER-mediated pathway to produce adipic acid via ER-mediated pathway, and the xylose-to-butanetriol pathway to produce 1,2,4- butanetriol. See Table 1. The heterologous pathways shown in Table 1 can also be introduced into yeast in the absence of the ED pathway but with enhanced production of iron-sulfur clusters in the same yeast.

[0207] Table 1 : Example pathways coupled to iron-sulfur cluster system that can metalate prokaryotic enzymes in yeast

[0208] As described herein, various approaches are designed in this disclosure to deliver ironsulfur cluster to enzymes in microorganisms. These microorganism can be employed in continuous biomanufacturing systems. For example, one approach is to introduce a prokaryotic iron-sulfur cluster utilizing pathway (Entner-Doudoroff (ED) pathway) that is known to be significantly more efficient (for biomanufacturing purposes) than the glycolysis pathway in yeast: the ED pathway uses 50% less ATP than glycolysis, reducing biomass production from about 50% to about 90%, as well as increasing the overall output. Since the ED pathway produces only one ATP, it can result in less energy directed towards biomass formation and a more efficient use of energy towards production. This has been shown by Zymomonas mobilis. which relies on the ED pathway and attains higher ethanol yields than SaccharomycesWSGR Docket No. 66551-702.601 cerevisiae1, as well as with yeast engineered with futile cycles to consume excess ATP that exhibit increased ethanol production3. This pathway can be coupled with additional iron-sulfur cluster containing pathways of commercial importance. These additional iron-sulfur cluster containing pathways have so far only been known to be expressed in prokaryotes but can provide additional value when expressed in yeast (Table 1). Metalation of heterologous enzymes containing iron-sulfur clusters is challenging in yeast due to the selective nature of yeast’s native iron-sulfur cluster delivery system. Methods to improve the metalation of prokaryotic enzymes may help implementing and optimizing novel high-value pathways for biomanufacturing. By coupling to a pathway as efficient as the ED pathway, and lowering flux to slow growth but maintaining the same output as in glycolysis, the overall stability of production can be increased in yeast. Therefore, the compositions and methods disclosed herein teaches a technical path to improve the iron-sulfur cluster delivery activity in yeast. In some embodiments, an exogenous ED pathway and one or more additional iron-sulfur cluster containing pathways are introduced into an organism, such as, for example, a yeast, in the presence of one or more exogenous ironsulfur producing systems.Culture media or condition

[0209] Provided herein are culture media. A culture medium may comprise at least a molecule for supporting growth of a cell. A culture medium may comprise at least a molecule for supporting growth of a cell in vitro or ex vivo. A culture medium may comprise a carbon source, a nitrogen source, a mineral source, a vitamin source, water, salt, oxygen, carbon dioxide, or a combination thereof.

[0210] In some embodiments, the culture media of yeast may comprise iron or sulfur source. The iron or sulfur source may comprise cysteine. In some embodiments, the concentration of cysteine in the culture is at about 10 mg / L, about 20 mg / L, about 30 mg / L, about 40 mg / L, about 50 mg / L, about 60 mg / L, about 70 mg / L, about 80 mg / L, about 90 mg / L, about 100 mg / L, about 110 mg / L, about 120 mg / L, about 130 mg / L, about 140 mg / L, about 150 mg / L, about 160 mg / L, about 170 mg / L, about 180 mg / L, about 190 mg / L, about 200 mg / L, about 210 mg / L, about 220 mg / L, about 230 mg / L, about 240 mg / L, about 250 mg / L, about 260 mg / L, about 270 mg / L, about 280 mg / L, about 290 mg / L, about 300 mg / L, about 310 mg / L, about 320 mg / L, about 330 mg / L, about 340 mg / L, about 350 mg / L, about 360 mg / L, about 370 mg / L, about 380 mg / L, about 390 mg / L, about 400 mg / L, about 410 mg / L, about 420 mg / L, about 430 mg / L, about 440 mg / L, about 450 mg / L, about 460 mg / L, about 470 mg / L, about 480 mg / L, about 490, or about 500 mg / L. In some embodiments, the concentration of cysteine in the culture is from about 10 to about 20 mg / L, from about 20 to about 30 mg / L, from about 30 to about 40 mg / L, from about 40 mg / L to about 50 mg / L, from about 50 mg / L to about 60 mg / L, from about 60 mg / L to about 70WSGR Docket No. 66551-702.601 mg / L, from about 70 mg / L to about 80 mg / L, from about 860 mg / L to about 90 mg / L, from about 90 mg / L to about 100 mg / L, from about 100 mg / L to about 110 mg / L, from about 110 mg / L to about 120 mg / L, from about 120 mg / L to about 130 mg / L, from about 130 mg / L to about 140 mg / L, from about 140 mg / L to about 150 mg / L, from about 150 mg / L to about 160 mg / L, from about 160 mg / L to about 170 mg / L, from about 170 mg / L to about 180 mg / L, from about 180 mg / L to about 190 mg / L, from about 190 mg / L to about 200 mg / L, from about 200 mg / L to about 210 mg / L, from about 210 mg / L to about 220 mg / L, from about 220 mg / L to about 230 mg / L, from about 230 mg / L to about 240 mg / L, from about 240 mg / L to about 250 mg / L, from about 250 mg / L to about 260 mg / L, from about 260 mg / L to about 270 mg / L, from about 270 mg / L to about 280 mg / L, from about 280 mg / L to about 290 mg / L, from about 290 mg / L to about 300 mg / L, from about 300 mg / L to about 310 mg / L, from about 310 mg / L to about 320 mg / L, from about 320 mg / L to about 330 mg / L, from about 330 mg / L to about 340 mg / L, from about 340 mg / L to about 350 mg / L, from about 350 mg / L to about 360 mg / L, from about 360 mg / L to about 370 mg / L, from about 370 mg / L to about 380 mg / L, from about 380 mg / L to about 390 mg / L, from about 390 mg / L to about 400 mg / L, from about 400 mg / L to about 410 mg / L, from about 410 mg / L to about 420 mg / L, from about 420 mg / L to about 430 mg / L, from about 430 mg / L to about 440 mg / L, from about 440 mg / L to about 450 mg / L, from about 450 mg / L to about 460 mg / L, from about 460 mg / L to about 470 mg / L, from about 470 mg / L to about 480 mg / L, from about 480 mg / L to about 490, or from about 490 mg / L to about 500 mg / L. In some embodiments, the concentration of cysteine in the culture is no more than 10 mg / L, no more than 20 mg / L, no more than 30 mg / L, no more than 40 mg / L, no more than 50 mg / L, no more than 60 mg / L, no more than 70 mg / L, no more than 80 mg / L, no more than 90 mg / L, no more than 100 mg / L, no more than 110 mg / L, no more than 120 mg / L, no more than 130 mg / L, no more than 140 mg / L, no more than 150 mg / L, no more than 160 mg / L, no more than 170 mg / L, no more than 180 mg / L, no more than 190 mg / L, no more than 200 mg / L, no more than 210 mg / L, no more than 220 mg / L, no more than 230 mg / L, no more than 240 mg / L, no more than 250 mg / L, no more than 260 mg / L, no more than 270 mg / L, no more than 280 mg / L, no more than 290 mg / L, no more than 300 mg / L, no more than 310 mg / L, no more than 320 mg / L, no more than 330 mg / L, no more than 340 mg / L, no more than 350 mg / L, no more than 360 mg / L, no more than 370 mg / L, no more than 380 mg / L, no more than 390 mg / L, no more than 400 mg / L, no more than 410 mg / L, no more than 420 mg / L, no more than 430 mg / L, no more than 440 mg / L, no more than 450 mg / L, no more than 460 mg / L, no more than 470 mg / L, no more than 480 mg / L, no more than 490, or no more than 500 mg / L.

[0211] In some embodiments, the culture media of yeast may comprise iron or sulfur source. The iron or sulfur source may comprise a ferrous salt or Fe(II) salt. In some embodiments, theWSGR Docket No. 66551-702.601Fe(II) salt added together with cysteine includes but is not limited to: FeCh, FeBr2, Feb, Fe(NOs)2, FeSCh, ferrous gluconate, ferrous fumarate, or ferrous succinate, etc.

[0212] In some embodiments, the concentration of ferrous salt in the culture is at about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, about 300 pM, about 310 pM, about 320 pM, about 330 pM, about 340 pM, about 350 pM, about 360 pM, about 370 pM, about 380 pM, about 390 pM, or about 400 pM. In some embodiments, the concentration of ferrous salt in the culture is from about 10 to about 20 pM, from about 20 to about 30 pM, from about 30 to about40 pM, from about 40 pM to about 50 pM, from about 50 pM to about 60 pM, from about 60 pM to about 70 pM, from about 70 pM to about 80 pM, from about 860 pM to about 90 pM, from about 90 pM to about 100 pM, from about 100 pM to about 110 pM, from about 110 pM to about 120 pM, from about 120 pM to about 130 pM, from about 130 pM to about 140 pM, from about 140 pM to about 150 pM, from about 150 pM to about 160 pM, from about 160 pM to about 170 pM, from about 170 pM to about 180 pM, from about 180 pM to about 190 pM, from about 190 pM to about 200 pM, from about 200 pM to about 210 pM, from about 210 pM to about 220 pM, from about 220 pM to about 230 pM, from about 230 pM to about 240 pM, from about 240 pM to about 250 pM, from about 250 pM to about 260 pM, from about 260 pM to about 270 pM, from about 270 pM to about 280 pM, from about 280 pM to about 290 pM, from about 290 pM to about 300 pM, from about 300 pM to about 310 pM, from about 310 pM to about 320 pM, from about 320 pM to about 330 pM, from about 330 pM to about 340 pM, from about 340 pM to about 350 pM, from about 350 pM to about 360 pM, from about 360 pM to about 370 pM, from about 370 pM to about 380 pM, from about 380 pM to about 390 pM, or from about 390 pM to about 400 pM. In some embodiments, the concentration of ferrous salt in the culture is no more than 10 pM, no more than 20 pM, no more than 30 pM, no more than40 pM, no more than 50 pM, no more than 60 pM, no more than 70 pM, no more than 80 pM, no more than 90 pM, no more than 100 pM, no more than 110 pM, no more than 120 pM, no more than 130 pM, no more than 140 pM, no more than 150 pM, no more than 160 pM, no more than 170 pM, no more than 180 pM, no more than 190 pM, no more than 200 pM, no more than 210 pM, no more than 220 pM, no more than 230 pM, no more than 240 pM, no more than 250 pM, no more than 260 pM, no more than 270 pM, no more than 280WSGR Docket No. 66551-702.601 pM, no more than 290 pM, no more than 300 pM, no more than 310 pM, no more than 320 pM, no more than 330 pM, no more than 340 pM, no more than 350 pM, no more than 360 pM, no more than 370 pM, no more than 380 pM, no more than 390 pM, or no more than 400 pM. The ferrous salt can be any one of FeCh, FeBr2, Feb, Fe(NOs)2, FeSCU, ferrous gluconate, ferrous fumarate, or ferrous succinate.

[0213] A culture medium may have a volume of greater than or equal to about 1 nanoliter (nL), 10 nL, 100 nL, 1 microliter (pL), 10 pL, 100 pL, 1 milliliter (mL), 10 mL, 100 mL, 1 liter (L), 10 L, lxlOA2 L, lxl0A3 L, lxlOA4 L or more. A culture medium may have a volume of less than or equal to about 1 nL, 10 nL, 100 nL, 1 pL, 10 pL, 100 pL, 1 mL, 10 mL, 100 mL, 1 L, 10 L, lxlOA2 L, lxl0A3 L, or lxlOA4 L.

[0214] A culture medium may have a pH of greater than or equal to about 0.25, 0.5, 0.75, 1,1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25,6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11,11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, or 14. A culture medium may have a pH of less than or equal to about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3,3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25,8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, or 14.

[0215] A culture medium may have a temperature of greater than or equal to about: -20 °C, -10 °C, -4 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45°C, 46°C, 47 °C, 48 °C, 49 °C, 50 °C or more. A culture medium may have a temperature of less than or equal to about: -20 °C, -10 °C, -4 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45°C, 46°C, 47 °C, 48 °C, 49 °C, or 50 °C.

[0216] The culture condition may comprise aerobic condition. The culture condition may comprise anaerobic condition. The culture condition may comprise microaerobic condition. The culture condition may comprise greater than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 % or more oxygen (v / v) of the total atmosphere. The culture condition may comprise microaerobicWSGR Docket No. 66551-702.601 condition. The culture condition may comprise less than or equal to about: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, or 50 % oxygen (v / v) of the total atmosphere.

[0217] All terms are intended to be understood as they can be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0218] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0219] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0220] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0221] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0222] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may meanWSGR Docket No. 66551-702.601 within an acceptable error range for the particular value, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0223] “Aerobic conditions” refers to environmental conditions wherein oxygen (O2) is present in any quantity during growth of the engineered organism. “Anaerobic conditions” refers to environmental conditions wherein no detectible oxygen is present during growth of the engineered organism. “Microaerobic conditions” refers to environmental conditions wherein oxygen (O2) is present at no more than 5% (v / v) of the total atmosphere during growth of the engineered organism.

[0224] As used herein the term “endogenous,” when used in reference to a polynucleotide, a gene, or a polypeptide generally refers to a native polynucleotide or gene in its natural location in the genome of an organism, or a native polypeptide, that is transcribed and translated from this location in the genome.

[0225] As used herein the term “exogenous,” when used in reference to a polynucleotide, a gene, or a polypeptide generally refers a protein sequence or a nucleic acid sequence that is utilized in an organism other than the one from which it originated.

[0226] As used herein the term “heterogeneous” generally refers to a subject or entity from a different origin. For example, when used in connection with a polypeptide, gene, or gene product present in a particular cell or organism, the term is such that the relevant polypeptide, gene, or gene product is 1) manipulated by human hands. 2) Introduced into a cell or organism (or precursor thereof) through human hands, and / or 3) naturally into the associated cell or organism (e.g., associated cell type or organism). Reveal that it is not produced or does not exist in the state.

[0227] As used herein the term “transformation” generally refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance with or without selections. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

[0228] As used herein the term of an “isolated” polypeptide or a fragment, variant, or derivative thereof generally refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cellsWSGR Docket No. 66551-702.601 are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.EXAMPLESExample 1. Delivered iron-sulfur clusters efficiently to nitrogenase in E. coli

[0229] Provided herein are methods for generating an engineered organism comprising Fe-S cluster biosynthesis pathway and nitrogenase. The Fe-S cluster is delivered to the nitrogenase of the engineered organism.

[0230] In E. coli, the iron-sulfur cluster biogenesis proceeds through either the housing keeping ISC (iron-sulfur cluster assembly) pathway or the stress-induced SUF (sulfur formation) pathway for the viability of the organism. On the one hand, E. coil's canonical iron-sulfur cluster ISC may not be sufficient to deliver iron-sulfur clusters. On the other hand, E. coil's SUF system can deliver iron-sulfur clusters to nitrogenase in A", coli organisms, even in organisms with different nitrogenase systems, such as, for example, Paenibacillus polymyxa (“Pp”) (FIG. 2) and Klebsiella oxytoca (“Ko”) (FIG. 3).

[0231] Abbreviations used regarding various inducible iron-sulfur cluster delivery systems:• SUF from E. coli (“Ec”)• SufCB fusion only from Blastocystis (“B sp”)• nifUS from Entamoeba histolytica (“Eh”)• nifUS from Klebsiella (“Ko”)• SUF from Monocercomonoides exilis (“Me”)• The iron-sulfur cluster carrier protein (“mrp”) from Desulfovibrio vulgaris Hildenborough (“DvH”)

[0232] FIG. 2 shows nitrogenase activities in E. coli, with nifH from Paenibacillus polymyxa expressed heterologously with various inducible iron-sulfur cluster delivery systems. The native nitrogenase is nif from Paenibacillus polymyxa and nifF / nifl from Klebsiella. The inducible cluster delivery systems added include: SUF from E. coli, nifUS from Klebsiella, SufCB fusion only from Blastocystis, nifUS from Entamoeba histolytica, and mrp from DvH. SUF from E. coli seems to induce nitrogenase activities better than any other inducible cluster delivery system tested. Nitrogenase was tested via the acetylene reduction assay (ARA), where the ability of nitrogenase to reduce acetylene is used a proxy for nitrogen fixation. See Temme K, Zhao D, Voigt CA. “Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca.” Proc Natl Acad Sci U S A. 2012 May l;109(18):7085-90, which is incorporated by reference in its entirety. This activity is measured either in whole cells or in crude extracts. In both cases the cells or extracts are added to a vial, the vial is capped and 10% acetylene by volume is added. AtWSGR Docket No. 66551-702.601 specified times, head space samples are taken and measured in a Gas Chromatograph. For crude extracts, since cells are lysed, we added an adenosine triphosphate (ATP) generating system, as well as a reductant, to provide energy and reducing power to nitrogenase, respectively.

[0233] Many methods can be used to introduce a nucleic acid molecule into yeast. For example, transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, Gietz et al., 1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991, Methods in Enzymology 194: 182-7, each of which references is incorporated herein by reference in its entirety.

[0234] In some embodiments, the integration of a gene of interest into a yeast microorganism can be accomplished according to the principle of homologous recombination. An integration cassette containing a module comprising a yeast marker gene and / or the gene of interest to be integrated (“sequence to be inserted”) is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (“recombinogenic sequences”). After transforming the yeast with the cassette by an appropriate method, a homologous recombination between the recombinogenic sequences may result in the sequence to be inserted replacing the chromosomal region in between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette. See, Orr-Weaver et al., 1981, PNAS USA 78: 6354-58, which is incorporated herein by reference in its entirety.

[0235] In some embodiment, the integration cassette for integration of a gene of interest into a yeast microorganism may include the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome. In some embodiments, the heterologous gene includes a non-native gene encoding an iron-sulfur cluster producing system. The selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic sequences can be chosen depending on the desired integration site suitable for the desired application. In some embodiments, integration of a gene into the chromosome of the yeast microorganism may occur via random integration. See, Kooistra et al., 2004, Yeast 21 : 781-792, which is incorporated herein by reference in its entirety.

[0236] The exogenous nucleic acid molecule contained within a yeast cell of the present disclosure can be maintained within that yeast cell in any form. For example, the exogenous nucleic acid molecule can be integrated into the genome of the cell or maintained in an episomal state that can stably be passed on (“inherited”) to its daughter cells. Such extra-chromosomal genetic elements (such as plasmids, mitochondrial genome, etc.) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover,WSGR Docket No. 66551-702.601 the yeast cells can be stably or transiently transformed. In addition, the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described herein.

[0237] FIG. 3 shows nitrogenase activities in E. coli, with nifH from Klebsiella with various inducible iron-sulfur cluster delivery systems. The native nitrogenase is nif from Klebsiella. The inducible cluster delivery systems added include: SufCB fusion only from Blastocystis, SufADSE from E. coli, nifUS from Entamoeba histolytica, SufABCDSE from E. coli, mrp from DvH, and nifUS from Klebsiella. SufABCDSE from E. coli seems to induce nitrogenase activities better than any other inducible cluster delivery system tested.

[0238] Based on the data shown in FIG. 2 and FIG. 3, different iron-sulfur delivery systems may behave differently in different bacteria. For example, adding the “missing” component to B sp SUF from E. coli SUF may improve nitrogenase activity in Paenibacillus polymyxa cluster but not in Klebsiella cluster. The iron-sulfur cluster carrier protein (“mrp”) from Desulfovibrio vulgaris Hildenborough does not display a measurable improvement in nitrogenase activity in E. coli, but it may display an improvement in yeast. Eh NifUS displays lower activity in E. coli than Klebsiella nifUS does. A mixture of different iron-sulfur cluster systems were used to find whether there is an optimal system for delivery. Some of the mixtures exhibited higher nitrogenase activity than ISC.

[0239] In some cases, the heterologous polypeptide may not have the full SUF system as present in Ec and Me. For example, as described herein, the heterologous polypeptide may not comprise SufD that can decrease the Fe-S activity of another heterologous polypeptide (see, for example, FIG. 10)Example 2. Delivery of iron-sulfur clusters in yeast

[0240] Provided herein are methods for generating an engineered organism comprising Fe-S cluster biosynthesis. The Fe-S cluster is delivered to the heterologous polypeptide of the engineered organism. The engineered organism is eukaryote. The eukaryote is yeast.

[0241] Previous research shows that yeast may not deliver iron-sulfur clusters to prokaryotic enzymes. The canonical iron-sulfur clusters delivery system in yeast is found in the mitochondria. This system may only deliver iron-sulfur clusters specifically to yeast proteins and typically does not properly metalate heterologously expressed enzymes in the yeast cytosol.

[0242] In this experiment activity of EDD enzyme in yeast were tested. The results show that iron-sulfur clusters were delivered to prokaryotic enzymes expressed in yeast (up to 60%). See FIG. 4. In this case, the activities of 6-phosphogluconate dehydratase (EDD) were measured in yeast cells (Saccharomyces cerevisiae) with or without cysteine desulfurase (IscS) and / or other iron-sulfur clusters producing eukaryotic (Blastocystis; Bsp) or prokaryotic (E. coli) systemsWSGR Docket No. 66551-702.601(see for example, FIG. 10). IscS can make iron-sulfur clusters in vitro. In this experiment purified IscS was added to the yeast cells and helped understand what percentage of the EDD enzyme was in apo-form. In FIG. 4, ED by itself means no additional iron-sulfur cluster system is added. In this experiment iron-sulfur cluster delivery system was tested for its ability to metalate a heterologously expressed EDD enzyme (from E. coll) in the yeast cytosol in vivo. To determine the fraction of EDD enzyme metalated, the iron-sulfur cluster was reconstituted and delivered in vitro by the addition of IscS (purified from E. coli) and EDD activities measured before and after in vitro metalation were compared.

[0243] Increased EDD activity was observed in the presence of high concentrations of exogenous cysteine and iron in the culture in the wildtype yeast comprising EDD activities. See FIG. 5. In this experiment, the addition of iron (200 pM, Fe(II) salt) and cysteine (300 mg / L) significantly facilitated this activity. The backgrounds were various yeast mutants that have been published to import more iron. Even with these various yeast mutants, the presence of SUF (from E. coli) in the system was shown to boost EDD activities even higher. The addition of both iron and cysteine was shown to facilitate higher activity. See FIG. 5. While this study used 200 pM iron and 300 mg / L cysteine, other concentrations or ranges of concentrations of iron or cysteine, respectively, are possible. In this study the prokaryotic enzyme expressed / delivered in yeast was EDD. Other prokaryotic enzymes are possible, including, for example, NifH.

[0244] While the canonical systems introduced into yeast were not sufficient to metalate NifH, our modified SUF system, with SufCB from B.sp and SufASE (from E. coli) was able to do so as seen in FIG. 10

[0245] The experiment in FIG. 10 was to evaluate different changes in the SUF system to see if they can result in higher activity and thus better metalation and delivery of iron-sulfur clusters, in this example with NifH from nitrogenase. The right-hand side are ARA assay controls (activity of Klebsiella cluster in E. coli, using NifUS). The middle graphs are reconstituted activity: this is where in vitro we added the full E. coli SUF system from crude extract to assay how much of the activity was reconstituted (measured as 100 % activity in this experiment). This activity was lower than the one on the right because of the Cl NifH used here. Cl NifH is a NifH protein generated from consensus sequence that is soluble in Yeast. Klebsiella NifH is not soluble in yeast. Cl NifH has activity with Klebsiella NifDK (purified from E. coli for this experiment) but had lower activity potentially due to compatibility issues (since NifH directly binds to NifDK).

[0246] Referring to data on the left side: While Bsp. SufCB showed some activity, combining ‘missing’ proteins from the SUF operon (found in other organisms) can provide better metalation. Consistent with this notion, when proteins from E. coli were added, the activity didWSGR Docket No. 66551-702.601 not increase. One hypothesis was that SufD caused interference. After removing SufD (thus having SufCB from Bsp and SufASE from E. coli), significantly higher activity was observed. The right-hand side in FIG. 10 shows ARA assay controls (activity of Klebsiella cluster in E. coli, using NifUS).

[0247] In this experiment, the reconstituted activity with E. coli SUF system in lysate was added in vitro. This shows the maximal activity that can have been obtained. This system was able to obtain -33% of max activity (potentially by adjusting expression, and compatibility between SufCB from one organism and SufASE from another we can achieve closer to full activity).

[0248] FIG. 11 depicts the nitrogenase activities of nitrogenase Fe protein (nifH) expressed in yeast alongside various FeS systems. For Ec. suf high (vs. all other systems tested here), the expression of all suf genes in E coli was increased by expressing the genes on a high copy plasmid. For each yeast strain, nifH protein was purified and tested for activity using ARA assay as described herein. In this assay, each reaction consisted of nifH purified from various yeast strains, Klebsiella oxytoca nitrogenase MoFe protein (nifDK) purified from E. coli, reductant, ATP-generating components, and acetylene. In the presence of metalated, active nifH, nitrogenase reduces acetylene to ethylene. For the two rightmost data, the purified nifH protein was incubated with the lysate of an E coli strain expressing Ec sufABCDSE to reconstitute the [4Fe4S] cluster on nifH before to an acetylene reduction reaction.

[0249] Thus, the combination of Bsp sufCB with components from the Ec suf operon are effective at providing [4Fe4S] to nifH in vivo in yeast. Additionally, increasing the expression of our [4Fe4S] system improves metalation, indicating that the expression of the non-native suf system is driving the metalation of nifH (see Ec. suf High).

[0250] FIG. 12 depicts the nitrogenase Fe protein (nifH) expressed in yeast alongside genomically-integrated Ec suf (gsuf) or Ko nifUS on a plasmid (pUS). nifH protein was purified and then in vitro reconstituted by incubating with the lysate of an E coli strain expressing Ec sufABCDSE (r suf) or the lysate of an E coli strain expressing Ko nifUS (r_US). After the reconstitution reaction, nifH was purified again and then added to an in vitro ARA assay (as described herein) containing the reconstituted and re-purified nifH, Klebsiella oxytoca nitrogenase MoFe protein (nifDK) purified from E. coli, reductant, ATP-generating components, and acetylene. The activity of nifH was determined by measuring the amount of ethylene produced via the reduction of acetylene by the nitrogenase in the reaction. As a control (bottom data point), nifH was directly assayed for acetylene reduction without the reconstitution step.

[0251] Thus, the data shows that in the in vitro [4Fe4S] reconstitution of nifH, Ec suf is more effective than Ko nifUS.WSGR Docket No. 66551-702.601

[0252] FIG. 13 depicts the EDD enzyme activity when expressed in and purified from different yeast strains expressing EDD alongside various FeS systems. The leftmost bar (“none”) refers to a yeast strain that only expressed EDD without any additional FeS systems. The activity of the enzyme was assayed in vitro by measuring the formation of KDPG (as described elsewhere in this disclosure).

[0253] Thus, the data shows that the expression of any FeS system improves metalation of EDD enzyme expressed in yeast compared to EDD expressed alone. The combination of a subset of Ec suf proteins and Bsp sufCB is the most effective in this assay.

[0254] FIG. 14 depicts the EDD enzyme purified from different yeast strains expressing EDD alongside various FeS systems. The leftmost bar (“none”) refers to a yeast strain that only expressed EDD without any additional FeS systems. The activity of the enzyme was assayed in vitro by measuring the formation of KDPG (as described elsewhere in this disclosure). Two nifUS systems from different organisms were test and improve activity relative to the native FeS systems in yeast for EDD, but are less effective than the three suf systems from different organisms that were tested.

[0255] Thus, the data shows that the expression of any FeS system improves metalation of EDD enzyme expressed in yeast compared to EDD expressed alone.

[0256] FIG. 15 depicts the radical SAM enzyme nifB (nitrogenase cofactor biosynthesis protein) expressed in yeast alongside the full Ec.suf system and subsequently purified. To assay for protein activity (left side of graph), the purified protein was added to an acetylene reduction assay reaction, consisting of lysate of an E. coli strain containing Ko nif cluster with AnifB, molybdenum, SAM, homocitrate, Na2SO3, reductant, ATP -generating components, and acetylene. In the presence of active nifB, the nitrogenase reduces acetylene to ethylene, which can be quantified to report enzyme activity. As a negative control, lysate alone was added to an ARA reaction as well.

[0257] Thus, the data shows that NifB purified from yeast is active, indicating successful metalation in yeast.

[0258] In some embodiments, a combination of cysteine and ferrous salt is added to the culture to grow the yeast. In some embodiments, when adding a combination of cysteine and ferrous salt to the culture to grow the yeast, the cysteine is added in any of the concentrations disclosed above together with the ferrous salt of any kind disclosed above in any of the concentrations disclosed above.

[0259] Sulfur is used for the biogenesis of iron-sulfur cluster-containing proteins in vivo. Sulfur is a component of the iron-sulfur clusters that are incorporated into such proteins. Sulfur is also a component of glutathione, which is used in iron-sulfur cluster biogenesis in many organisms.WSGR Docket No. 66551-702.601Cysteine is one of the direct source of sulfur for these processes in many organisms. Increased cellular sulfur in the form of cysteine can be provided by increasing cellular uptake of exogenous cysteine.

[0260] In another experiment, EDD activities of different colonies of yeast cells were measured with or with iron and cysteine and / or other iron-sulfur clusters in wild-type (WT) background. As shown in FIG. 6, the addition of iron alone is not as effective as the addition of both iron and cysteine. B sp has better overall activity. If Me is used, activity can be higher. It is possible that a suf + eukaryotic aspect to explain why the activity in Me is better: suf system coming from eukaryotic organism works better in yeast (eukaryote).

[0261] FIG. 7 shows the effect of various mutants of yeast that improve iron delivery. In this experiment, iron (about 200 M) and cysteine (about 300 mg / L) were added to all yeast backgrounds. Various iron-sulfur systems were tested. The backgrounds were various yeast mutants that have been reported to import more iron. As shown in FIG. 7, different iron-sulfur clusters behave differently in the same yeast cells.

[0262] FIG. 8 shows the effect of different C-terminal tripeptide targeting complex recognition (TCR) tags on the delivery of iron-sulfur clusters to prokaryotic iron-sulfur enzymes (EDD) expressed in yeast. As shown in FIG. 8, the TCR tags do not provide iron-sulfur to EDD in yeast.

[0263] FIG. 9 shows improving natural iron-sulfur systems for yeast by, for example, adding modified suf systems. The results showed that Me suf activities can be improved by added E. coli SufA. While adding iron-sulfur components from E. coli to Me improved suf activity, similar effect was not observed in B sp. It is possible that this may be caused by the interference from E. coli SufD. Other combinations of SUF from E. coli without SufD may improve suf activities in B sp. In addition, Me SufDSU + B sp SufCB can be introduced with or without A. coli SufA in yeast.

[0264] References (each of which references is incorporated herein by reference in its entirety):1. Van Aalst, A. C. A. et al. Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production. Synth. Syst. Biotechnol. 7, 554-566 (2022).2. Zymomonas mobilis: an alternative ethanol producer - Panesar - 2006 - Journal of Chemical Technology & Biotechnology - Wiley Online Library. See scij oumal s. onlinelibrary .wiley . com / doi / ab s / 10.1002 / j ctb .1448.3. Zahoor, A., Messerschmidt, K., Boecker, S. & Klamt, S. ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production. Biotechnol. Biofuels 13, 185 (2020).WSGR Docket No. 66551-702.6014. Benisch, F. & Boles, E. The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase. J. Biotechnol. 171, 45-55 (2014).5. Biz, A. & Mahadevan, R. Overcoming Challenges in Expressing Iron-Sulfur Enzymes in Yeast. Trends Biotechnol. 39, 665-677 (2021).

[0265] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.NUMBERED EMBODIMENTS1. An engineered organism comprising: a nitrogenase; and one or more non-native iron-sulfur cluster producing systems; wherein the nitrogenase is expressed in a species of the engineered organism.2. The engineered organism of embodiment 1, wherein the species is klebsiella and / or paenibacillus.3. The engineered organism of embodiment 1 or 2, wherein the nitrogenase is a klebsiella nitrogenase or a paenibacillus nitrogenase.4. The engineered organism of any one of embodiments 1-3, wherein the engineered organism is A. coli.5. The engineered organism of embodiment 4, wherein the engineered organism is yeast.6. The engineered organism of embodiment 5, wherein the yeast is S. cerevisiae.7. The engineered organism of any one of embodiments 1-6, wherein the one or more nonnative iron-sulfur cluster producing systems comprise a sulfur formation (SUF) systemWSGR Docket No. 66551-702.601 or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough .8. The engineered organism of any one of embodiments 1-6, wherein the one or more nonnative iron-sulfur cluster producing systems comprise an eukaryotic iron-sulfur cluster producing system.9. The engineered organism of any one of embodiments 1-6, wherein the one or more nonnative iron-sulfur cluster producing systems comprise SUF from E. coh. SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, a full SUF system in E. coli, or a full SUF system in Monocercomonoides exilis, or a combination thereof.10. The engineered organism of any one of embodiments 1-6, wherein the one or more nonnative iron-sulfur cluster producing systems comprise SufCB fusion only from Blastocystis and one or more polypeptides from SUF from E. coli.11. The engineered organism of embodiment 10, wherein the engineered organism is an eukaryote of a different species from the Blastocystis .12. The engineered organism of any one of embodiments 1-11, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a portion of the nitrogenase.13. An engineered organism comprising: a non-native prokaryotic enzyme; and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic enzyme is expressed in a species of the engineered organism.14. The engineered organism of embodiment 13, wherein the engineered organism is yeast.15. The engineered organism of embodiment 14, wherein the yeast is S. cerevisiae.16. The engineered organism of embodiment 13, wherein the engineered organism is a plant.17. The engineered organism of any one of embodiments 13-16, wherein the non-native prokaryotic enzyme is an iron-sulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster.18. The engineered organism of any one of embodiments 13-17, wherein one of the one or more non-native iron-sulfur cluster producing systems comprises a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof.19. The engineered organism of any one of embodiments 13-17, wherein the one or more non-native iron-sulfur cluster producing systems comprises SUF from E. coli, SufCBWSGR Docket No. 66551-702.601 fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in E. coli, a full SUF system in E. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof.20. The engineered organism of any one of embodiments 13-17, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA.21. The engineered organism of embodiment 20, wherein the one or more non-native ironsulfur cluster producing systems comprise SufA from E. coli.22. The engineered organism of any one of embodiments 13-21, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB.23. The engineered organism of embodiment 22, wherein the one or more non-native ironsulfur cluster producing systems comprise SufB from E. coli.24. The engineered organism of any one of embodiments 13-23, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC.25. The engineered organism of embodiment 24, wherein the one or more non-native ironsulfur cluster producing systems comprise SufC from E. coli.26. The engineered organism of any one of embodiments 13-22, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB.27. The engineered organism of embodiment 26, wherein the one or more non-native ironsulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli.28. The engineered organism of any one of embodiments 13-27, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS.29. The engineered organism of embodiment 28, wherein the one or more non-native ironsulfur cluster producing systems comprises SufS from E. coli.30. The engineered organism of any one of embodiments 13-29, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE.31. The engineered organism of embodiment 30, wherein the one or more non-native ironsulfur cluster producing systems comprises SufE from E. coli.32. The engineered organism of any one of embodiments 13-31, wherein none of the one or more non-native iron-sulfur cluster producing systems comprises SufD from E. coli.33. The engineered organism of any one of embodiments 13-17, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in A’. coli.WSGR Docket No. 66551-702.60134. The engineered organism of any one of embodiments 13-716, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis.35. The engineered organism of any one of embodiments 13-17, wherein the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism.36. The engineered organism of embodiment 35, wherein the engineered organism is a second eukaryotic organism different from the eukaryotic organism.37. The engineered organism of any one of embodiments 13-36, wherein the one or more non-native iron-sulfur cluster producing systems are configured-produce more ironsulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine.38. The engineered organism of any one of embodiments 13-36, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a portion of the non-native prokaryotic enzyme.39. An engineered organism comprising: a non-native prokaryotic system of Entner-Doudoroff pathway (EDP); and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic system is expressed in a species of the engineered organism.40. The engineered organism of embodiment 39, wherein the engineered organism is yeast.41. The engineered organism of embodiment 40, wherein the yeast is S. cerevisiae.42. The engineered organism of any one of embodiments 39-41, wherein the non-native prokaryotic system comprises an iron-sulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster.43. The engineered organism of any one of embodiments 39-42, wherein one of the one or more non-native iron-sulfur cluster producing systems comprises a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof.44. The engineered organism of any one of embodiments 39-42, wherein the one or more non-native iron-sulfur cluster producing systems comprises SUF from E. coh. SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in A. coli, a full SUF system in A. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof.WSGR Docket No. 66551-702.60145. The engineered organism of any one of embodiments 39-42, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA.46. The engineered organism of embodiment 45, wherein the one or more non-native ironsulfur cluster producing systems comprise SufA from E. coli.47. The engineered organism of any one of embodiments 39-46, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB.48. The engineered organism of embodiment 47, wherein the one or more non-native ironsulfur cluster producing systems comprise SufB from E. coli.49. The engineered organism of any one of embodiments 39-48, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC.50. The engineered organism of embodiment 49, wherein the one or more non-native ironsulfur cluster producing systems comprise SufC from E. coli.51. The engineered organism of any one of embodiments 39-48, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB.52. The engineered organism of embodiment 51, wherein the one or more non-native ironsulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli.53. The engineered organism of any one of embodiments 39-52, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS.54. The engineered organism of embodiment 53, wherein the one or more non-native ironsulfur cluster producing systems comprises SufS from E. coli.55. The engineered organism of any one of embodiments 39-54, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE.56. The engineered organism of embodiment 55, wherein the one or more non-native ironsulfur cluster producing systems comprises SufE from E. coli.57. The engineered organism of any one of embodiments 39-56, wherein none of the one or more non-native iron-sulfur cluster producing systems comprises SufD from E. coli.58. The engineered organism of any one of embodiments 39-42, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in A. coli.59. The engineered organism of any one of embodiments 39-42, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis.60. The engineered organism of any one of embodiments 39-42, wherein the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism.WSGR Docket No. 66551-702.60161. The engineered organism of embodiment 60, wherein the engineered organism is a second eukaryotic organism different from the eukaryotic organism.62. -The engineered organism of any one of embodiments 39-61, wherein the one or more non-native iron-sulfur cluster producing systems are configured-produce more ironsulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine.63. The engineered organism of any one of embodiments 39-61, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a prokaryotic enzyme of the prokaryotic system.64. A composition, comprising: a. the engineered organism of any one of embodiments 1-63, b. iron; and c. cysteine.

Claims

WSGR Docket No. 66551-702.601CLAIMSWHAT IS CLAIMED IS:

1. An engineered organism comprising: a nitrogenase; and one or more non-native iron-sulfur cluster producing systems; wherein the nitrogenase is expressed in a species of the engineered organism.

2. The engineered organism of claim 1, wherein the species is klebsiella and / or paenibacillus.

3. The engineered organism of claim 1, wherein the nitrogenase is a klebsiella nitrogenase or a paenibacillus nitrogenase.

4. The engineered organism of claim 1, wherein the engineered organism is E. coli.

5. The engineered organism of claim 4, wherein the engineered organism is yeast.

6. The engineered organism of claim 5, wherein the yeast is S. cerevisiae.

7. The engineered organism of claim 1, wherein the one or more non-native iron-sulfur cluster producing systems comprise a sulfur formation (SUF) system or an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough.

8. The engineered organism of claim 1, wherein the one or more non-native iron-sulfur cluster producing systems comprise an eukaryotic iron-sulfur cluster producing system.

9. The engineered organism of claim 1, wherein the one or more non-native iron-sulfur cluster producing systems comprise SUF from E. coli, SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, a full SUF system in E. coli, or a full SUF system in Monocercomonoides exilis, or a combination thereof.

10. The engineered organism of claim 1, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufCB fusion only from Blastocystis and one or more polypeptides from SUF from E. coli.

11. The engineered organism of claim 10, wherein the engineered organism is an eukaryote of a different species from the Blastocystis.

12. The engineered organism of claim 1, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metal ate at least a portion of the nitrogenase.

13. An engineered organism comprising: a non-native prokaryotic enzyme; and one or more non-native iron-sulfur cluster producing systems;WSGR Docket No. 66551-702.601 wherein the prokaryotic enzyme is expressed in a species of the engineered organism.

14. The engineered organism of claim 13, wherein the engineered organism is yeast.

15. The engineered organism of claim 14, wherein the yeast is S. cerevisiae.

16. The engineered organism of claim 13, wherein the engineered organism is a plant.

17. The engineered organism of claim 13, wherein the non-native prokaryotic enzyme is an iron-sulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster.

18. The engineered organism of claim 13, wherein one of the one or more non-native ironsulfur cluster producing systems comprises a sulfur formation (SUF) system or an ironsulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof.

19. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises SUF from E. coif SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in E. coli, a full SUF system in E. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof.

20. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA.

21. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA from E. coli.

22. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB.

23. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB from E. coli.

24. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC.

25. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC from E. coli.

26. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB.

27. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli.WSGR Docket No. 66551-702.60128. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS.

29. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS from E. coli.

30. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE.

31. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE from E. coli.

32. The engineered organism of claim 13, wherein none of the one or more non-native ironsulfur cluster producing systems comprises SufD from E. coli.

33. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system incoli.

34. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis.

35. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism.

36. The engineered organism of claim 35, wherein the engineered organism is a second eukaryotic organism different from the eukaryotic organism.

37. The engineered organism of claim 13, wherein the one or more non-native iron-sulfur cluster producing systems are configured-produce more iron-sulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine.

38. The engineered organism of claim 13, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a portion of the non-native prokaryotic enzyme.

39. An engineered organism comprising: a non-native prokaryotic system of Entner-Doudoroff pathway (EDP); and one or more non-native iron-sulfur cluster producing systems; wherein the prokaryotic system is expressed in a species of the engineered organism.

40. The engineered organism of claim 39, wherein the engineered organism is yeast.

41. The engineered organism of claim 40, wherein the yeast is S. cerevisiae.

42. The engineered organism of claim 39, wherein the non-native prokaryotic system comprises an iron-sulfur cluster protein whose functions depend on an expression or assembly of an iron-sulfur cluster.WSGR Docket No. 66551-702.60143. The engineered organism of claim 39, wherein one of the one or more non-native ironsulfur cluster producing systems comprises a sulfur formation (SUF) system or an ironsulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, or a combination thereof.

44. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises SUF from E. coif SufCB fusion only from Blastocystis, NifUS from Entamoeba histolytica, NifUS from Klebsiella, NifUS from E. his, NifUS from K. oxy, an iron-sulfur cluster carrier protein (mrp) from Desulfovibrio vulgaris Hildenborough, SufADSE in E. coli, a full SUF system in E. coli, SufDSUBC in Monocercomonoides exilis, or a full SUF system in Monocercomonoides exilis, or a combination thereof.

45. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA.

46. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufA from E. coli.

47. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB.

48. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufB from E. coli.

49. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC.

50. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise SufC from E. coli.

51. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC and SufB.

52. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprise a fusion protein comprising SufC from E. coli and SufB from E. coli.

53. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS.

54. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufS from E. coli.

55. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE.WSGR Docket No. 66551-702.60156. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises SufE from E. coli.

57. The engineered organism of claim 44, wherein none of the one or more non-native ironsulfur cluster producing systems comprises SufD from E. coli.

58. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system incoli.

59. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems comprises a full SUF system in Monocercomonoides exilis.

60. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems are from an eukaryotic organism.

61. The engineered organism of claim 60, wherein the engineered organism is a second eukaryotic organism different from the eukaryotic organism.

62. The engineered organism of claim 39, wherein the one or more non-native iron-sulfur cluster producing systems are configured-produce more iron-sulfur clusters when supplemented with iron and cysteine than when not supplemented with iron and cysteine.

63. The engineered organism of claim 39, wherein iron-sulfur clusters produced by the one or more non-native iron-sulfur cluster producing systems are configured-metalate at least a prokaryotic enzyme of the prokaryotic system.

64. A composition, comprising: a. the engineered organism of any one of claims 1-63, b. iron; and c. cysteine.

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