Methods and engineered microorganisms for the production of HEME biosynthetic pathway products

Abstract

The present invention relates to methods for producing heme comprising culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium and converting the secreted coproporphyrin III and / or coproheme III into heme, as well as to genetically engineered microorganisms capable of secreting uroporphyrin III, uroporphyrin I, coproporphyrin III and / or coproheme III.

Description

METHODS AND ENGINEERED MICROORGANISMS FOR THE PRODUCTION OF HEME BIOSYNTHETIC PATHWAY PRODUCTS

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to US Provisional Application No. 63 / 602,463 filed November 24, 2023.

[0003] FIELD

[0004] The present disclosure relates to methods and genetically engineered microorganisms for the production of heme biosynthetic pathway products.

[0005] BACKGROUND

[0006] Porphyrins and their precursors find applications in the healthcare, pharmaceutical, and food industry. The demand for natural plant-based porphyrins is increasing especially with the increased popularity of plant-based meat alternatives. Therefore, large-scale production of these compounds is of commercial interest. Currently, most porphyrins are produced chemically. These chemical methods might not be effective and environmentally friendly, and often result in low yields with no selective production of specific porphyrin isomers. Bio-based production can offer a potential solution to overcome these limitations. However, product toxicity, ineffective conversion, low yield, and use of expensive feedstock, currently limit the industrial application of bio-based production.

[0007] SUMMARY

[0008] The present disclosure describes methods and genetically engineered microorganisms for the production of heme biosynthetic pathway products, such as heme.

[0009] The present disclosure provides a two-step method for the production of heme comprising: (a) culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium; and (b) converting the secreted coproporphyrin III and / or coproheme III into heme.

[0010] Further provided herein is a genetically engineered microorganism capable of secreting uroporphyrin III, uroporphyrin I, coproporphyrin III, coproporphyrin I, coproheme III and / or coproheme I.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying figures illustrate embodiments of the invention by way of example.

[0013] Figure 1. Exemplary heme biosynthetic pathway.

[0014] Figure 2. The tricarboxylic acid cycle and glyoxylate shunt.

[0015] Figure 3. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides (CPC-SbmA / c7 / ?A.st / / ?4 / pK-hemA). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers ofUP-I and UP-III. All values are reported as means±SD (n=2).

[0016] Figure 4. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides , and HemB from A’. coli (CPC-SbmA / c / AA.st / Ati / pK-hemAB). (A) Time profiles of 5- ALA and PBG biosynthesis. (B) Final extracellular titers of UP-I and UP-III. All values are reported as means±SD (n=2).

[0017] Figure 5. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemC from E. coli (CPC-SbmA / c / AA.st / Ati / pK-hem ABC). Time profiles of 5-ALA and PBG biosynthesis. All values are reported as means±SD (n=2).

[0018] Figure 6. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB from E. coli, and HemC from B. subtilis (CPC-SbmA / c / AA.st / Ati / pK- hemABCB). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers of UP-I and UP-III. All values are reported as means±SD (n=2).

[0019] Figure 7. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemD from E. coli (CPC-SbrnA / c / AAst / AU / pK-hemABD). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers of UP-I and UP-III. All values are reported as means±SD (n=2).

[0020] Figure 8. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB, HemD and HemE from A’. coli (CPC-SbmA / c / AA.st / AU / pK-hemABD-E). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers of UP-I, UP-III, CP- I and CP-III. All values are reported as means±SD (n=2).

[0021] Figure 9. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB, HemD and HemE from A’. coli (CPC-SbmA / c / AA.st / AU / pK-hemAB-DE). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers of UP-I, UP-III, CP- I and CP-III. All values are reported as means±SD (n=2).

[0022] Figure 10. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli (CPC-SbmA / c7 / ?A.st / Ati / pK-hemAB-E). (A) Timeprofiles of 5-ALA and PBG biosynthesis. (B) Final extracellular titers of UP -I, UP-III, CP-I and CP-III. All values are reported as means±SD (n=2).

[0023] Figure 11. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides , HemB, HemD and HemE from E. coli, and HemH from B. subtilis (CPC- SbmAzc7AA.st / A4 / pK-hem ABD-EFI). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular and intracellular titers of CP-I, CP-III, Fe-CP-I and Fe-CP-III. All values are reported as means±SD (n=2).

[0024] Figure 12. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli, and HemH from B. subtilis (CPC- SbmAzc7AA.st / A4 / pK-hemAB-EFI). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular and intracellular titers of CP-I, CP-III, Fe-CP-I and Fe-CP-III. All values are reported as means±SD (n=2).

[0025] Figure 13. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli, and HemH from B. subtilis (CPC- SbmAzc7AA.st / A4 / pI<-hemABE-H). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular and intracellular titers of CP-I, CP-III, Fe-CP-I and Fe-CP-III. All values are reported as means±SD (n=2).

[0026] Figure 14. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB, HemD and HemE from A’. coli, and HemH and HemQ from B. subtilis (CPC- SbmAzc7AA.S6 / A4 / pK-hemABD-EHQ). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular and intracellular titers of UP -I, UP-III, CP-I, CP-III, FeCP-III, PP-IX and heme. All values are reported as means±SD (n=2).

[0027] Figure 15. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli, and HemH and HemQ from B. subtilis (CPC- SbmAzc / 7?As,<7A4 / pK-hemAB-EHQ). (A) Time profiles of 5-ALA and PBG biosynthesis. (B) Final extracellular and intracellular titers of CP-I, CP-III, FeCP-I, FeCP-III, PP-IX and heme. All values are reported as means±SD (n=2).

[0028] Figure 16. Heme produced through coproporphyrin as opposed to protoporphyrin by introducing HemH and HemQ from gram positive bacteria into E. coli. (A) Final extracellular and intracellular titers of UP -I, UP-III, CP-I, CP-III and heme from E. coli expressing HemA from R. sphaeroides , HemB, HemD, HemE, HemF, HemG and HemH from A’. coli (CPC-SbmAzc / 7?As AA / pK-hemABD-EFGH). (B) Final extracellular and intracellular titers of UP-I, UP-III, CP-I, CP-III, Fe-CP-III, PP-IX and heme from E. coli expressing HemA from R.sphaeroides, HemB, HemD, and HemE from A’. colt, and HemH and HemQ from B. subtilis (CPC- SbmAzc / 7?As,<7A4 / pK-hemABD-EHQ).

[0029] Figure 17. Comparison of the UP -I, UP-III, CP-I, CP-III, Fe-CP-III, extracellular heme and intracellular heme yields from CPC-SbmAiclRAsdhA E. coli containing either pK- HemABD-E, pK-HemAB-E, pK-HemABD-EFGH or pK-HemABD-EHQ, wherein HemA is from R. sphaeroides , HemB, HemD, HemE, HemF, and HemG are from E. coli, and HemQ is from B. subtilis. HemH in pK-HemABD-EFGH is from A’. coli and HemH in pK-HemABD-EHQ is from B. subtilis. All values are reported as means±SD (n=2).

[0030] Figure 18. Bioreactor cultivation of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli, and HemH from B. subtilis (CPC- SbrnA / c / AAsv / AUpK-hemAB-EH). (A) Final extracellular and intracellular titers of CP-I, CP-III, Fe-CP-I and Fe-CP-III. All values are reported as means±SD (n=2). HPLC chromatograms showing absorbance at 400nm to visualize heme biosynthetic products from the CFM of E. coli expressing HemA, HemB, HemE and HemH treated with B. subtilis HemQ-his rich lysate (B) for 1 hour and (C) overnight.

[0031] Figure 19. HPLC chromatogram showing absorbance at 400nm to visualize heme biosynthetic products from the CFM of E. coli expressing HemA from R. sphaeroides, HemB and, HemE from E. coli, and HemH from B. subtilis (CPC-SbmAzc / AA.st / AU / pK-hemAB- EH) treated with purified B. subtilis HemQ-his overnight.

[0032] Figure 20. HPLC chromatogram showing absorbance at 400nm to visualize heme biosynthetic products from the CFM of E. coli expressing HemA from R. sphaeroides, HemB and HemE from E. coli, and HemH from B. subtilis (C PC-SbmA / c / AA.s / AUpK-hem ABE- H) treated with purified S. aureus HemQ-his overnight.

[0033] DETAILED DESCRIPTION

[0034] Heme has low solubility and is difficult for cells to secrete, leading to intracellular accumulation and toxicity, which may be the reason for low yields of biosynthetically produced heme. The present disclosure relates to the discovery that the yield of heme can be increased by culturing a microorganism that secretes coproporphyrin III and / or coproheme III into a culture medium and then converting the secreted coproporphyrin III and / or coproheme III into heme. This approach is demonstrated to result in coproporphyrin III yields that are much higher than intracellular heme yields (Figure 17). Furthermore, secreted coproheme III can be converted into heme extracellularly (Figures 18B-C, 19, 20) to achieve higher yields of heme than would be produced by conventional methods of intracellular heme production.

[0035] Accordingly, the present disclosure provides a method of producing heme, comprising: (a) culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium; and (b) converting the secreted coproporphyrin III and / or coproheme III into heme.

[0036] The present disclosure further provides a genetically engineered microorganism capable of secreting uroporphyrin III, uroporphyrin I, coproporphyrin III, coproporphyrin I, coproheme III and / or coproheme I.

[0037] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0038] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0039] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0040] As used here, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g, gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Altschul etal., 1997). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g, of XBLAST and NBLAST) can be used (see, e.g, the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0041] The sequences of the present disclosure may be at least 80% identical to the sequences described herein; in another example, the sequences may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical at the nucleic acid or amino acid level to sequences described herein. Importantly, proteins encoded by variant sequences retain the activity and specificity of the proteins encoded by the reference sequences.

[0042] Heme Biosynthetic Pathway Overview

[0043] Tetrapyrroles are natural, colorful, and essential metabolic pigment compounds produced in most living organisms. Tetrapyrroles differ in the oxidation state of their ring system, the chelated metal ion, and the ring substituents. Structurally, tetrapyrroles consist of four pyrrole rings linked in a cyclic (i.e., cyclic tetrapyrroles) or linear (i.e., linear tetrapyrroles) array. Porphyrins are the main class of cyclic tetrapyrroles found in nature with heme as a representative example. On the other hand, linear tetrapyrroles are normally derived from cyclic tetrapyrroles by opening the methine bridge linking the four pyrrole rings.

[0044] Heme is a porphyrin comprised of ferrous ion (Fe2+) and protoporphyrin-IX (PP- IX), and is essential for all cells. The structure of heme is shown below:

[0045] Biosynthesis of heme is highly conserved across biological kingdoms and the associated biosynthetic pathways, mechanisms, and enzymes have been well documented. The common precursor for biosynthesis of most tetrapyrroles including heme is 5-aminolevulinic acid (5-ALA).

[0046] Figure 1 illustrates an exemplary heme biosynthetic pathway: Two 5-ALA molecules condense to form porphobilinogen (PBG) by PBG Synthase (HemB). Four PBG molecules are subsequently polymerized by PBG deaminase (HemC) to form a linear tetrapyrrole hydroxymethylbilane (HMB). HMB undergoes spontaneous ring closure to form uroporphyrinogen I (UPG-I), and uroporphyrinogen III synthase (HemD) catalyzes HMB ring closure to form uroporphyrinogen III (UPG-III). Both UPG-I and UPG-III can be spontaneously oxidized to form uroporphyrin I (UP-I) and uroporphyrin III (UP-III), respectively, as metabolic end products. Alternatively, UPG-I / III are enzymatically converted to coproporphyrinogen I / III (CPG-I / III) by uroporphyrinogen III decarboxylase (HemE). CPG-I / III can also spontaneously oxidize to make the end products coproporphyrin I / III (CP-I / III). Here, heme biosynthesis splits into two distinct pathways: classical (protoporphyrin-dependent; PPD) and non-classical (coproporphyrin-dependent; CPD). Portions of the PPD and CPD branches might coexist in an organism, leading to different mechanisms for heme biosynthesis under different environmental conditions and challenges.

[0047] In the PPD pathway, coproporphyrinogen III is decarboxylated and oxidized to form protoporphyrinogen by coproporphyrinogen III oxidase (HemF) and then protoporphyrinogen is oxidized to PP-IX by protoporphyrinogen oxidase (HemG). PP-IX is chelated by ferrochelatase (HemH) to form heme. The CPD pathway differs in the order of the last three reactions. Rather than decarboxylation of the propionate groups around the ring, the iron ion is inserted into CP-III first by ferrochelatase (HemH) to form coproheme III (Fe-CP-III). Coproheme III is then decarboxylated to form heme by iron-coproporphyrin oxidative decarboxylase (HemQ). There is not an analogous enzyme in the PPD pathway.

[0048] As used herein, the term “heme biosynthetic pathway products” refers to any compounds that are end products or intermediates of the heme biosynthetic pathway or derivatives thereof (such as those produced by oxidation), including the porphyrins shown in Figure 1. As used herein, the term “heme biosynthetic pathway enzyme” refer to any enzyme involved in producing or modifying end products or intermediates of the heme biosynthetic pathway, including the enzymes indicated in Figure 1.

[0049] Heme Biosynthetic Pathway Precursors

[0050] The common precursor for biosynthesis of most tetrapyrroles is 5-ALA, which can be derived via either of the two unrelated metabolic routes of the C5 and C4 pathways. These two pathways differ in only the first 5-ALA-generating step, all subsequent reactions toward heme are identical. The C5 pathway, which is found in most bacteria and all archaea / plants, is initiated by the ligation of glutamate and tRNAGluvia glutamyl-tRNA synthase (GluTS or HemA) and subsequent reduction via glutamyl-tRNA reductase (GluTR) to form glutamate-l-semialdehyde (GSA). Then, GSA is transaminated by glutamate- 1 -semial dehyde-2,1 -aminomutase (GSAM) to generate 5-ALA. On the other hand, in the C4 pathway (also known as the Shemin pathway) which is found in humans, animals, fungi, and certain proteobacteria, 5-ALA is synthesized in a one-step reaction catalyzed by 5-ALA synthase (ALAS or HemA) by molecular fusion of succinyl-CoA and glycine.

[0051] Many bacteria use the C5 pathway to constitutively synthesize heme. Hence, many bacteria can serve as feasible hosts for further engineering to derive heme-overproducing strains. However, as a-ketoglutarate, a key precursor of the C5 pathway for heme biosynthesis is readily consumed as an intermediate of the oxidative tricarboxylic acid (TCA) cycle under aerobic conditions, diverting carbon flux from the TCA cycle toward heme biosynthesis via this C5 compound can be challenging. Therefore, in some embodiments, the C4 pathway with succinyl- CoA as the precursor is used, with metabolic strategies diverting carbon flux away from the TCA cycle via this C4 compound.

[0052] Formation of succinate / succinyl-CoA in some microorganisms, such as Escherichia coli, can be achieved via three oxygen-dependent pathways, namely, (i) reductive TCA branch (i.e., reverse TCA cycle), (ii) oxidative TCA cycle, and (iii) glyoxylate shunt (Figure 2). Normally, succinate accumulates as an end-product of mixed acid fermentation via the reductive TCA branch which is activated under anaerobic conditions. Although the reductive TCA branch can potentially yield high-level succinate, this pathway is generally unfavorable as it is limited by the availability of reducing equivalents. Under aerobic conditions, succinate is normally used up as an intermediate of the oxidative TCA cycle without accumulation, except for the conditions of oxidative stress and / or acetate / fatty-acid consumption under which succinate can be aerobically derived via operational glyoxylate shunt. Glyoxylate shunt can provide anaplerotic reactions and bypass the CO2-producing steps in the oxidative TCA cycle, thereby conserving carbon atoms for the cell. Since succinyl-CoA is involved in the central metabolism in a complex manner and its formation can be mediated through multiple oxygen-sensitive pathways, manipulation of the carbon flux around this C4 compound can be challenging. Therefore, proper operation of the TCA cycle can play a major role in directing carbon flux toward the C4 pathway for tetrapyrrole biosynthesis.

[0053] Genetic Alterations

[0054] The present disclosure provides for a genetically engineered microorganism capable of secreting UP -III, UP -I, CP-III, CP-I, Fe-CP-III and / or Fe-CP-I. As used herein, the term “genetically engineered” and its derivatives refer to a microorganism whose genetic material has been altered using molecular biology techniques known in the art, such as but not limited to molecular cloning, recombinant deoxyribonucleic acid (DNA) methods, transformation and gene transfer. The genetically engineered microorganism includes a living modified microorganism, genetically modified microorganism or a transgenic microorganism. Genetic alterations include, but are not limited to, addition, deletion, substitution, modification and / or mutation of genetic material, that can result in inactivation of select genes on the host genome (e.g., gene knockout), reduction of the expression of selected genes on the host genome (e.g., gene knock-down), overexpression of select genes / operons using expression plasmids (e.g., heterologous gene expression), and, insertion of select genes into the host genome (e.g., gene knock-in).

[0055] As used herein, the term “functional inactivation” or its derivatives, includes alterations which may result in: i) a protein with decreased or absent functional activity when compared to the wild-type protein; ii) expression of a truncated protein with decreased or absent functional activity; or iii) the deletion or inactivation of the gene encoding the protein.

[0056] The phrase “introducing a nucleic acid molecule into a microorganism” includes both the stable integration of the nucleic acid molecule into the genome of a microorganism to prepare a genetically engineered microorganism as well as the transient integration of the nucleic acid into microorganism. The introduction of a nucleic acid into a cell is also known in the art as transformation. The nucleic acid molecules may be introduced into the microorganism using techniques known in the art including, without limitation, agitation with glass beads, electroporation, agrobacterium-mediated transformation, an accelerated particle delivery method, i.e., particle bombardment, a cell fusion method or by any other method to deliver the nucleic acid vectors to a microorganism.

[0057] The term "nucleic acid molecule" or its derivatives, as used herein, is intended to include unmodified DNA or ribonucleic acid (RNA) or modified DNA or RNA. For example, it is useful for the nucleic acid molecules of the disclosure to be composed of single- and doublestranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and doublestranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, it is useful for the nucleic acid molecules to be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. In some embodiments, the genetically engineered microorganism comprises at least one nucleic acid molecule described herein.

[0058] As used herein, the term “exogenous” refers to an element that has been introduced into a cell. An exogenous element can include a protein or a nucleic acid. An exogenous nucleic acid is a nucleic acid that has been introduced into a cell, such as by a method of transformation. An exogenous nucleic acid may code for the expression of an RNA and / or a protein. An exogenous nucleic acid may have been derived from the same species (homologous) or from a different species (heterologous). An exogenous nucleic acid may comprise a homologous sequence that is altered such that it is introduced into the cell in a form that is not normally found in the cell in nature. For example, an exogenous nucleic acid that is homologous may contain mutations, being operably linked to a different control region, or being integrated into a different region of the genome, relative to the endogenous version of the nucleic acid. An exogenous nucleic acid maybe incorporated into the chromosomes of the transformed cell in one or more copies, into the plastid or mitochondrial DNA of the transformed cell, or be maintained as a separate nucleic acid outside of the transformed cell genome.

[0059] The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0060] Nucleic acid sequences as described herein can be provided in nucleic acid molecules in different arrangements or combinations. As used herein with reference to nucleic acid molecules that encode a gene or polypeptide, the term “at least one exogenous nucleic acid molecule encoding” or “at least one nucleic acid molecule encoding” means that all encoded sequences are collectively comprised in one or more nucleic acid molecules. In some embodiments, all encoded sequences are comprised in separate nucleic acid molecules, all encoded sequences are comprised in the same nucleic acid molecule, or combinations where some encoded sequences are comprised in separate nucleic acid molecules and some encoded sequences are comprised together in the same nucleic acid molecule. For example, in one embodiment wherein a microorganism comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemE, HemH and HemQ, nucleic acid sequences encoding a HemA and HemB can be provided together in a first nucleic acid molecule, nucleic acid sequences encoding a HemE and HemH can be provided in a second nucleic acid molecule, and a nucleic acid sequence encoding a HemQ can be provided in a third nucleic acid molecule. Alternatively, sequences that encode all of the enzymes of a heme biosynthetic pathway can be provided together in the same nucleic acid molecule. Where more than one sequence that encodes an enzyme is provided in the same nucleic acid molecule, the sequences can be provided in separate expression cassettes, or together in the same expression cassette. Where two or more sequences are in the same expression cassette, they may form a polycistronic mRNA for subsequent translation, wherein each of the sequences has its own ribosomal binding site for translation. Where two or more sequences are in the sameexpression cassette, they can be provided in the same open reading frame so as to produce a fusion protein. Two or more sequences that encode a fusion protein can be separated by linker sequences that encode restriction nuclease recognition sites or self-cleaving peptide linkers. Accordingly, a genetically modified microorganism for the production of heme biosynthetic pathway products can be engineered by stepwise transfection with multiple nucleic acid molecules that each comprise nucleic acid sequences that encode one or more enzymes of a heme biosynthetic pathway, or with a single nucleic acid molecule that comprises nucleic acid sequences that encode multiple or all of the enzymes of a heme biosynthetic pathway. Nucleic acid molecules encoding genes or polypeptides can include, without limitation, plasmids, artificial chromosomes, episomes, and expression cassettes inserted into known vectors or inserted into chromosomes or plastid or mitochondrial DNA.

[0061] As used herein, the term “vector” or “nucleic acid vector” is a type of nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a microorganism. The transgenic DNA can encode a heterologous protein, which can be expressed in and isolated from a microorganism. The transgenic DNA can be integrated into nuclear, mitochondrial or chloroplastic genomes through homologous or non-homologous recombination. The transgenic DNA can also replicate without integrating into nuclear, mitochondrial or chloroplastic genomes in an extra-chromosomal vector. The vector can contain a single, operably -linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence. Vectors useful in the present methods are well known in the art. In some embodiments, the exogenous nucleic acid molecule is a vector comprising a nucleic acid sequence encoding one or more heme biosynthetic pathway enzymes. In some embodiments, the exogenous nucleic acid molecules comprise vectors each comprising one or more nucleic acid sequences encoding one or more heme biosynthetic pathway enzymes.

[0062] The term “operably-linked”, as used herein, refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. For example, a transcriptional regulatory sequence or a promoter is operably-linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. The skilled person can readily recognize aspects of the transcription process, which include, but are not limited to, initiation, elongation, attenuation and termination. In general, an operably -linked transcriptional regulatory sequence joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.

[0063] As used herein, the term “tag” refers to an amino acid sequence that is recognized by an antibody. The tag amino acid sequence links to, for example, sequence of an enzyme, thereby allowing detection or isolation of the enzyme by the binding between the tag and the tag-specific antibody. For example, common tags known in the art include His, MYC, FLAG, V5, HA and HSV. These tags are useful when positioned at the N- or C-terminus. In some embodiments, the at least one exogenous nucleic acid molecule comprises one or more tag sequences. In some embodiments, the at least one exogenous nucleic acid molecule comprises one or more His tag sequences. In some embodiments, a His tag sequence is added to the N- or C- terminus of a heme biosynthetic pathway enzyme. In some embodiments, a His tag sequence is added to the N- or C- terminus of HemQ and / or HernH.

[0064] The word “expression” as used herein refers to the translation of a polypeptide (for example a protein of interest) encoded by a nucleic acid (such as an exogenous nucleic acid) and / or transcription of an RNA transcript (for example, mRNA or an interfering RNA, such as microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA) or Dicer-substrate smallinterfering RNA (DsiRNA)) encoded by a nucleic acid (such as an exogenous nucleic acid). As used herein, “increased expression” refers to an increase in the amount of translated polypeptide and / or RNA transcript encoded by a nucleic acid (such as an exogenous nucleic acid) in a cell. The increased expression may be relative to a wild-type microorganism.

[0065] Heme Biosynthetic Pathway Enzymes

[0066] In addition to the exemplary heme biosynthetic pathway shown in Figure 1, alternative enzymes can be used in a heme biosynthetic pathway in a genetically engineered microorganism. For example, in addition to the enzymes found in Escherichia coli, alternative enzymes of a heme biosynthetic pathway may be found in other bacteria (e.g., Rhodobacter, Synechocystis and / or Bacillis). Enzymes that differ in structure, but perform the same function, may be used interchangeably in a heme biosynthetic pathway in a genetically engineered microorganism of the present disclosure.

[0067] In addition to the wild-type enzymes found in organisms discussed herein, modified variants of these enzymes can be used in a heme biosynthetic pathway in a genetically engineered microorganism. Variants of enzymes for use in a heme biosynthetic pathway can be generated by altering the nucleic acid sequence encoding said enzyme to, for example, increase / decrease the activity of a domain, add / remove a domain, add / remove a signaling sequence, or to otherwise alter the activity or specificity of the enzyme.

[0068] In some embodiments, the microorganism is engineered to express or overexpress one or more enzymes of the heme biosynthesis pathway.

[0069] The hemA gene encodes HemA. In some embodiments, with respect to the C5 pathway, HemA refers to a glutamyl-tRNA reductase which reduces glutamyl-tRNA to form glutamate-1 -semialdehyde, by oxidizing NADPH into NADP+. In some embodiments, with respect to the C4 pathway, HemA refers to an aminolevulinic acid synthase which catalyzes the formation of 5-ALA from succinyl-CoA and glycine, releasing CoA and carbon dioxide in the process. In some embodiments, HemA is HemA from Rhodobacter sphaeroides . In some embodiments, HemA has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:1. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemA.

[0070] The hemB gene encodes HemB, an aminolevulinic acid dehydratase, also known as porphobilinogen synthase, a metalloenzyme that asymmetrically condenses two molecules of 5- ALA to form a porphobilinogen molecule and two water molecules. In some embodiments, HemB is HemB from E. coli. In some embodiments, HemB has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:2. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemB.

[0071] The hemC gene encodes HemC, a porphobilinogen deaminase, also known as hydroxymethylbilane synthase or uroporphyrinogen I synthase, which catalyzes the head to tail condensation of four porphobilinogen to form the linear tetrapyrrole hydroxymethylbilane. In some embodiments, HemC is HemC from E. coli. In some embodiments, HemC is HemC from Bacillus subtilis. In some embodiments, HemC has the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemC. In some embodiments, wherein the microorganism is E. coli, the microorganism does not comprise an exogenous nucleic acid molecule encoding HemC. In some embodiments, wherein the microorganism is E. coli, the microorganism comprises an exogenous nucleic acid molecule encoding HemC from B. subtilis.

[0072] The hemD gene encodes HemD, a uroporphyrinogen III synthase, also known as hydroxymethylbilane hydrolase, which catalyzes the asymmetrical cyclization of hydroxymethylbilane into uroporphyrinogen III. In some embodiments, HemD is HemD from E. coli. In some embodiments, HemD has the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO: 5. In someembodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemD. In some embodiments, the microorganism does not comprise an exogenous nucleic acid molecule encoding HemD.

[0073] The hemE gene encodes HemE, a uroporphyrinogen III decarboxylase, which decarboxylates the four acetic acid chains of uroporphyrinogen III to form coproporphyrinogen III. In some embodiments, HemE is HemE from E. coli. In some embodiments, HemE has the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:6. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemE.

[0074] The hemF gene encodes HemF, an aerobic coproporphyrinogen III oxidase, which oxidizes coproporphyrinogen III into protoporphyrinogen IX.

[0075] The hemG gene encodes HemG, a protoporphyrinogen oxidase, which oxidizes protoporphyrinogen IX into protoporphyrin IX.

[0076] The hemY gene encodes HemY, a protoporphyrinogen oxidase, also known as protox, which oxidizes coproporphyrinogen III into coproporphyrin III. In some embodiments, coproporphyrinogen III spontaneously oxidizes into coproporphyrin III. In some embodiments, HemY is HemY from E. coli. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemY.

[0077] The hemH gene encodes HemH, a ferrochelatase, which catalyzes the insertion of ferrous iron into coproporphyrin III to form coproheme III. Ferrochelatase also catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. In some embodiments, HemH is HemH from E. coli. In some embodiments, HemH is HemH from a gram-positive bacterium. In some embodiments, HemH is HemH from Bacillus subtilis. In some embodiments, HemH is HemH from Staphylococcus aureus. In some embodiments, HemH has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemH. In some embodiments, the endogenous HemH is functionally inactivated.

[0078] The hemQ gene encodes HemQ, an iron-coproporphyrin oxidative decarboxylase, also known as hydrogen peroxide-dependent heme synthase, which catalyzes the decarboxylation of coproheme III to form heme. . In some embodiments, HemQ is HemQ from a gram-positive bacterium. In some embodiments, HemQ is HemQ from B. subtilis. In some embodiments, HemQ is HemQ from Staphylococcus aureus. In some embodiments, HemQ has the amino acid sequenceof SEQ ID NO: 10 or SEQ ID NO: 11, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding HemQ. In some embodiments, the endogenous HemQ is functionally inactivated.

[0079] In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding at least one, two, three, four, five, six, seven, or eight of HemA, HemB, HemC, HemD, HemE, HemY, HemH and HemQ. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding at least one, two, three, four, five, or six of HemA, HemB, HemC, HemD, HemE, and HemH. In some embodiments, the microorganism is further engineered such that the endogenous hemH and / or hemQ gene is functionally inactivated.

[0080] In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA and HemB. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemC. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemD. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC and HemD.

[0081] In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemE. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA and HemE. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemB and HemE. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD and HemE. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemE and HemH. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemE and HemH. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemB, HemE and HemH. In some embodiments, themicroorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, HemE and HemH. In some embodiments, the microorganism is engineered to express or overexpress at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, HemE and HemH. In some embodiments, wherein the microorganism is not E. coli, the at least one exogenous nucleic acid molecule further encodes HemC.

[0082] Other Enzymes

[0083] In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to express or overexpress a protein which increases the production of a precursor molecule and / or the microorganism is engineered to functionally inactivate or decrease the expression of a protein which limits the production of a precursor molecule.

[0084] In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to increase production of succinyl-CoA when compared to a corresponding wild-type microorganism. In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to increase constitutive carbon flux through the glyoxylate cycle compared to a corresponding wild-type microorganism. In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to increase expression of an enzyme of the glyoxylate cycle, increase expression of an activator of the glyoxylate cycle, and / or decrease expression or functional inactivation of a repressor of the glyoxylate cycle.

[0085] The aceA gene encodes AceA, an isocitrate lyase, which catalyzes the cleavage of isocitrate to succinate and glyoxylate. In some embodiments, the AceA is AceA from E coli. In some embodiments, the expression of AceA is increased compared to the corresponding wild-type microorganism. In some embodiments, the constitutive expression of AceA in the microorganism is increased compared to the corresponding wild-type microorganism. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding AceA. In some embodiments, the constitutive expression of AceA in the microorganism is increased compared to the corresponding wild-type microorganism and the microorganism comprises an exogenous nucleic acid molecule encoding AceA.

[0086] The aceB gene encodes AceB, a malate synthase, which uses glyoxylate, acetyl- CoA and water to form malate and CoA. In some embodiments, the AceB is AceB from E. coli. In some embodiments, the expression of AceB is increased compared to the corresponding wildtype microorganism. In some embodiments, the constitutive expression of AceB in the microorganism is increased compared to the corresponding wild-type microorganism. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding AceB. In some embodiments, the constitutive expression of AceB in the microorganism is increased compared to the corresponding wild-type microorganism and the microorganism comprises an exogenous nucleic acid molecule encoding AceB.

[0087] The aceK gene encodes AceK, an isocitrate dehydrogenase kinase / phosphatase, which is a bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase. In some embodiments, the expression of AceK is increased compared to the corresponding wildtype microorganism. In some embodiments, the constitutive expression of AceK in the microorganism is increased compared to the corresponding wild-type microorganism. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding AceK. In some embodiments, the constitutive expression of AceK in the microorganism is increased compared to the corresponding wild-type microorganism and the microorganism comprises an exogenous nucleic acid molecule encoding AceK.

[0088] In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism has increased expression of isocitrate lyase, malate synthase and / or isocitrate dehydrogenase kinase / phosphatase. In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism has increased constitutive expression of isocitrate lyase, malate synthase and / or isocitrate dehydrogenase kinase / phosphatase, compared to the corresponding wild-type microorganism. In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism has increased constitutive expression of the aceBAK operon compared to the corresponding wild-type microorganism.

[0089] As used herein, the term “activator” refers to a protein which increases expression of one or more genes. In some embodiments, one or more activators of the glyoxylate cycle have increased expression compared to the corresponding wild-type microorganism.

[0090] In some embodiments, an activator of the glyoxylate cycle is fructose repressor(FruR). In some embodiments, the FruR acts as an activator for the transcription of isocitrate lyase,malate synthase and isocitrate dehydrogenase kinase / phosphatase. In some embodiments, the FruR acts as an activator for the transcription of the aceBAK operon. In some embodiments, the FruR is FruR from E. coli. In some embodiments, the expression of FruR is increased compared to the corresponding wild-type microorganism. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding FruR.

[0091] In some embodiments, an activator of the glyoxylate cycle is the integration host factor (IHF) protein. In some embodiments, the IHF acts as an activator for the transcription of isocitrate lyase, malate synthase and isocitrate dehydrogenase kinase / phosphatase. In some embodiments, the IHF acts as an activator for the transcription of the aceBAK operon. In some embodiments, the IHF is IHF from E. coli. In some embodiments, the expression of IHF is increased compared to the corresponding wild-type microorganism. In some embodiments, the microorganism comprises an exogenous nucleic acid molecule encoding IHF.

[0092] As used herein, the term “repressor” refers to a protein which decreases or inhibits expression of one or more genes. In some embodiments, one or more repressors of the glyoxylate cycle have decreased expression compared to the corresponding wild-type microorganism and / or are functionally inactivated.

[0093] In some embodiments, a repressor of the glyoxylate cycle is isocitrate lyase regulator (IcIR). IcIR represses the transcription of isocitrate lyase, malate synthase and isocitrate dehydrogenase kinase / phosphatase. In some embodiments, IcIR represses the transcription of the aceBAK operon. In some embodiments, the IcIR is IcIR from E. coli. In some embodiments, the expression of IcIR is decreased compared to the corresponding wild-type microorganism. In some embodiments, IcIR is functionally inactivated.

[0094] In some embodiments, a repressor of the glyoxylate cycle is aerobic respiration control protein (ArcA). ArcA is a global regulator which regulates genes involved in a variety of metabolic pathways. In some embodiments, the ArcA is ArcA from E. coli. In some embodiments, the expression of ArcA is decreased compared to the corresponding wild-type microorganism. In some embodiments, ArcA is functionally inactivated.

[0095] The sdhABCD genes encodes SdhABCD, the succinate dehydrogenase complex, which catalyzes the oxidation of succinate to fumarate by reducing ubiquinone to ubiquinol. In some embodiments, the SdhABCD is SdhABCD from E. coli. In some embodiments, the expression of SdhABCD is decreased compared to the corresponding wild-type microorganism. In some embodiments, SdhABCD is functionally inactivated.

[0096] The IdhA gene encodes LdhA, a lactate dehydrogenase A, which catalyzes the conversion of pyruvate into lactate by oxidizing NADH to NAD. In some embodiments, LdhA is LdhA from E. coli. In some embodiments, the expression of LdhA is decreased compared to the corresponding wild-type microorganism. In some embodiments, LdhA is functionally Inactivated.

[0097] In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to increase expression of malate synthase, isocitrate lyase, isocitrate dehydrogenase kinase / phosphatase, FruR, and / or IHF; and / or decrease expression or functional inactivation of IcIR, ArcA, SdhABCD, and / or LdhA. In some embodiments, in addition to expressing, overexpressing or functionally inactivating one or more of the heme biosynthetic pathway enzymes described above, the microorganism is further engineered to increase expression of AceB, AceA, AceK, FruR, and / or IHF; and / or decrease expression or functional inactivation of IcIR, ArcA, SdhABCD, and / or LdhA.

[0098] Types of microorganism

[0099] It would be appreciated by a skilled person that any suitable microorganism can be utilized herein. In some embodiments, the microorganism is a bacterium or a fungus.

[0100] In some embodiments, the bacterium is from the genera Alcaligenes, Arthr abaci er, Anaerobiospirillum, Bacillus, Brevibacterium, Corynebacterium, Enterococcus, Erwinia, Escherichia, Gluconobacter, Lactobacillus, Mannheimia, Paenibacillus, Rhizobium, Rhodobacter, Rhodococcus, Staphylococcus, Streptomyces, Synechocystis , and Zymomonas.

[0101] In some embodiments, the bacterium is a species of the class Gammoproteobacteria. In some embodiments, the bacterium is a species of the order Enterobacterales. In some embodiments, the bacterium is a species of the family Enterobacteriaceae. In some embodiments, the bacterium is a species of the genus Escherichia. In some embodiments, the bacterium is Escherichia coli. In some embodiments, the bacterium is a species of the class Bacilli. In some embodiments, the bacterium is a species of the order Bacillales. In some embodiments, the bacterium is a species of the family Bacillaceae. In some embodiments, the bacterium is a species of the genus Bacillus. In some embodiments, the bacterium is Bacillus subtilis. In some embodiments, the bacterium is Corynebacterium glutamicum.

[0102] In some embodiments, the microorganism is a fungus, for example from the genera Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Toruplosis. In some embodiments, the microorganism is Saccharomyces cerevisiae. In some embodiments, the microorganism is Pichia pastoris.

[0103] Methods

[0104] The present disclosure provides methods of producing heme, comprising: (a) culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium; and (b) converting the secreted coproporphyrin III and / or coproheme III into heme. Both naturally occurring microorganisms and genetically modified microorganisms capable of secreting coproporphyrin III and / or coproheme III can be used in the methods of the present invention.

[0105] As used herein, the word “secretes” and its derivatives, refer to the ability of the microorganism to produce and discharge a compound or polypeptide from inside the microorganism into the surrounding environment. In some embodiments, the microorganism secretes one or more heme biosynthetic pathway products. In some embodiments, the microorganism secretes UP-III and / or UP-I. In some embodiments, the microorganism secretes CP-III, CP-I, Fe-CP-III and / or Fe-CP-I. In some embodiments, the microorganism secretes CP- III, CP-I, Fe-CP-III and / or Fe-CP-I into the culture medium. In some embodiments, the microorganism secretes a heme biosynthetic pathway enzyme. In some embodiments, a microorganism secretes HemH and / or HemQ. In some embodiments, the microorganism secretes HemH and / or HemQ into the culture medium.

[0106] In some embodiments, the coproporphyrin III and / or coproheme III is converted into heme in the culture medium. In some embodiments, the culture medium comprises HemQ. In some embodiments, the culture medium comprises HemQ and HemH.

[0107] In some embodiments, step (b) comprises adding HemQ to the culture medium. In some embodiments, step (b) comprises adding HemQ and HemH to the culture medium.

[0108] As used herein, the word “isolated” and its derivatives, refer to the separation of specific compounds from a more complex matrix. Many methods for isolation are known in the art and are suitable for use in the present invention, including solvent extraction, chromatography and crystallization. In some embodiments, coproporphyrin III and / or coproheme III is isolated from the culture medium.

[0109] In some embodiments, step (b) comprises isolating the secreted coproporphyrin III and contacting the isolated coproporphyrin III with a solution comprising HemH or HemH / HemQ. In some embodiments, step (b) comprises isolating the secreted coproheme III and contacting the isolated coproheme III with a solution comprising HemQ.

[0110] As used herein, the word “co-culturing” and its derivatives, refer to the culturing of two or more microorganisms that secrete different compounds and / or polypeptides together. Furthermore, the microorganisms may be from the same or a different species. In someembodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is cocultured with a second microorganism that secretes HemQ. In some embodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is co-cultured with a second microorganism that secretes HemQ and HemH. In some embodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is co-cultured with a second microorganism that secretes HemQ and a third microorganism that secretes HemH.

[0111] In some embodiments, step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a second microorganism that secretes HemQ. In some embodiments, step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a second microorganism that secretes HemQ and HemH. In some embodiments, step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a second microorganism that secretes HemQ and a third microorganism that secretes HemH. In some embodiments, the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the HemQ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0112] In some embodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is a bacterium or fungus as described herein. In some embodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is a genetically engineered organism as described herein. In some embodiments, the microorganism that secretes coproporphyrin III and / or coproheme III is a genetically engineered bacteria or fungi as described herein.

[0113] In some embodiments, the genetically engineered microorganism described herein is capable of secreting at least 1 mg / L, at least 2 mg / L, at least 3 mg / L, at least 4 mg / L, at least 5 mg / L, at least 10 mg / L, at least 25 mg / L, at least 50 mg / L, at least 75 mg / L, at least 100 mg / L, at least 125 mg / L, at least 150 mg / L, at least 175 mg / L, or at least 200 mg / L of uroporphyrin III, uroporphyrin I, coproporphyrin III, coproporphyrin I, coproheme III, and / or coproheme I after 120 hours of cell culture.

[0114] Cell culture

[0115] Microorganisms may be cultured in conditions that are permissive to their growth. Common sources of fixed carbon that are used include glycerol, glucose, ethanol, or acetate. Microorganisms such as bacteria and fungi may be cultured using methods and conditions knownin the art, including shake-flask and bioreactor cultivation. Many media for the culture of microorganisms are known in the art and are suitable for use in the present invention including, but not limited to, lysogeny broth, super broth, M9 medium or yeast peptone dextrose broth.

[0116] In the C4 pathway, 5-ALA is synthesized from succinyl-CoA and glycine. To avoid having glycine limit this reaction, glycine can be added into the cell medium during the culturing process. In some embodiments, glycine is added into the cell medium during the culturing process. In some embodiments, glycine is added into the cell medium approximately 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 hours after inoculation. In some embodiments, approximately 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25 or 3.50 g / L of glycine is added into the cell medium during the culturing process. In some embodiments, approximately 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25 or 3.50 g / L of glycine is added into the cell medium approximately 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 hours after inoculation. In some embodiments, approximately 2 g / L of glycine is added into the cell medium approximately 30 hours after inoculation.

[0117] Embodiments

[0118] Particular embodiments of the invention include, without limitation, the following:1. A method of producing heme, comprising:(a) culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium; and(b) converting the secreted coproporphyrin III and / or coproheme III into heme.2. The method of paragraph 1, wherein the coproporphyrin III and / or coproheme III is converted into heme in the culture medium.3. The method of paragraph 2, wherein the culture medium comprises HemQ.4. The method of paragraph 3, wherein the culture medium further comprises HemH.5. The method of paragraph 1, wherein step (b) comprises adding HemQ to the culture medium.6. The method of paragraph 5, wherein step (b) further comprises adding HemH to the culture medium.7. The method of paragraph 1, wherein step (b) comprises isolating the secreted coproporphyrin III and / or coproheme III and contacting the isolated coproporphyrin III and / or coproheme III with a solution comprising HemQ.8. The method of paragraph 7, wherein the solution comprising HemQ further comprises HemH.9. The method of paragraph 1, wherein step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a second microorganism that secretes HemQ.10. The method of paragraph 9, wherein the second microorganism further secretes HemH.11. The method of paragraph 9, wherein step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a third microorganism that secretes HemH.12. The method of any one of paragraphs 1 to 11, wherein the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO: 8, or SEQ ID NO: 9.13. The method of any one of paragraphs 1 to 12, wherein the HemQ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NOTO or SEQ ID NOT E14. The method of any one of paragraphs 1 to 13, wherein the microorganism that secretes coproporphyrin III and / or coproheme III is from the genera Alcaligenes, Arthrobacter, Anaerobiospirillum, Aspergillus, Bacillus, Brevibacterium, Candida, Corynebacterium, Enterococcus, Erwinia, Escherichia, Fusarium, Gluconobacter, Hansenula, Kluyveromyces, Lactobacillus, Mannheimia, Paenibacillus, Pichia, Rhizobium, Rhodobacter, Rhodococcus, Saccharomyces, Schizosaccharomyces, Staphylococcus, Streptomyces, Synechocystis,, Toruplosis, or Zymomonas,15. The method of paragraph 14, wherein the microorganism that secretes coproporphyrin III and / or coproheme III is Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and / or Picchia pastoris.16. The method of any one of paragraphs 1 to 13, wherein the microorganism that secretes coproporphyrin III and / or coproheme III is a genetically engineered microorganism.17. The method of paragraph 16, wherein the genetically engineered microorganism is a bacterium.18. The method of paragraph 17, wherein the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, HemD, HemE, HemH, HemQ or combinations thereof.19. The method of paragraph 17, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemB; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemC; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemD; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, and HemD.20. The method of paragraph 17, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemE; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB and HemE; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, and HemE; or(vi) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE and HernH; or(vii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemE, and HernH; or(viii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB, HemE, and HernH; or(ix) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemE, and HernH; or(x) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, HemE, and HernH.21. The method of any one of paragraphs 18-20, wherein the bacterium is E. coli, and wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemC.22. The method of any one of paragraphs 18-20, wherein the bacterium is E. coli, and wherein the at least one exogenous nucleic acid molecule further encodes the HemC of B. subtilis.23. The method of any one of paragraphs 18-22, wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemD.24. The method of paragraph 20, wherein the bacterium is not E. coli, and wherein the at least one exogenous nucleic acid molecule further encodes HemC.25. The method of paragraph 18, wherein:(i) the endogenous hemH gene is functionally inactivated; and / or(ii) the endogenous hemQ gene is functionally inactivated.26. The method of any one of paragraphs 18 to 25, wherein the bacterium has increased production of succinyl-CoA compared to a corresponding wild-type microorganism.27. The method of any one of paragraphs 18 to 26, wherein the bacterium has increased constitutive carbon flux through the glyoxylate cycle compared to a corresponding wild-type microorganism.28. The method of any one of paragraphs 18 to 27, wherein the bacterium has increased constitutive expression of the aceBAK operon compared to a corresponding wild-type microorganism.29. The method of any one of paragraphs 18 to 28, wherein the bacterium has increased expression of an enzyme of the glyoxylate cycle, increased expression of an activator of the glyoxylate cycle, and / or decreased expression or functional inactivation of a repressor of the glyoxylate cycle.30. The method of paragraph 29, wherein:(i) the enzyme of the glyoxylate cycle is a malate synthase (e.g. AceB) and / or an isocitrate lyase (e.g. AceA); and / or(ii) the activator of the glyoxylate cycle is FruR and / or IHF; and / or(iii) the repressor of the glyoxylate cycle is IclR and / or ArcA; and / or(iv) iclR and / or arcA has a reduced expression or is functionally inactivated; and / or(v) sdhA, sdhB, sdhC, and / or sdhD has a reduced expression or is functionally inactivated; and / or(vi) IdhA has a reduced expression or is functionally inactivated.31. The method of any one of paragraphs 17 to 30, wherein the bacterium is from the genera Alcaligenes, Arthrobacter, Anaerobiospirillum, Bacillus, Brevibacterium, Corynebacterium, Enterococcus, Erwinia, Escherichia, Gluconob acter, Lactobacillus, Mannheimia, Paenibacillus, Rhizobium, Rhodobacter, Rhodococcus, Staphylococcus, Streptomyces, Synechocystis , and Zymomonas.32. The method of any one of paragraphs 17 to 31, wherein the bacterium is Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum.33. The method of paragraph 17, wherein the bacterium is E. coli, wherein the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE, and wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemC.34. The method of paragraph 33, wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemD.35. The method of paragraph 33 or 34, wherein iclR and sdhA are functionally inactivated.36. The method of any one of paragraphs 33 to 35, wherein IdhA is functionally inactivated.37. The method of paragraph 16, that is a genetically engineered yeast.38. The method of paragraph 37, wherein the yeast is from the genera Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Toruplosis.39. The method of paragraph 38, wherein the yeast is Saccharomyces cerevisiae or Pichia pas tor is.40. The method of any one of paragraphs 18 to 39, wherein the HemA comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:1.41. The method of any one of paragraphs 18 to 40, wherein the HemB comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:2.42. The method of any one of paragraphs 18 to 41, wherein the HemC comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:3 or SEQ ID NO:4.43. The method of any one of paragraphs 18 to 42, wherein the HemD comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:5.44. The method of any one of paragraphs 18 to 43, wherein the HemE comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:6.45. The method of any one of paragraphs 18 to 44, wherein the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.46. The method of any one of paragraphs 16 to 45, which is capable of secreting at least 1 mg / L, at least 2 mg / L, at least 3 mg / L, at least 4 mg / L, or at least 5 mg / L of coproporphyrin III and / or coproheme III after 120 hours of cell culture.47. A genetically engineered microorganism capable of secreting uroporphyrin III, uroporphyrin I, coproporphyrin III and / or coproheme III.48. The genetically engineered microorganism of paragraph 47, that is a genetically engineered bacterium.49. The genetically engineered microorganism of paragraph 48, wherein the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, HemD, HemE, HemH, HemQ or combinations thereof.50. The genetically engineered microorganism of paragraph 48, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemB; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemC; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemD; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, and HemD.51. The genetically engineered microorganism of paragraph 48, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemE; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB and HemE; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, and HemE; or(vi) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE and HemH; or(vii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemE, and HemH; or(viii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB, HemE, and HemH; or(ix) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemE, and HemH; or(x) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, HemE, and HemH.52. The genetically engineered microorganism of any one of paragraphs 49-51, wherein the bacterium is E. coli, and wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemC.53. The genetically engineered microorganism of any one of paragraphs 49-51, wherein the bacterium is E. coli, and wherein the at least one exogenous nucleic acid molecule further encodes the HemC of B. subtilis.54. The genetically engineered microorganism of any one of paragraphs 49-53, wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemD.55. The genetically engineered microorganism of paragraph 51, wherein the bacterium is not E. coli, and wherein the at least one exogenous nucleic acid molecule further encodes HemC.56. The genetically engineered microorganism of paragraph 49, wherein:(i) the endogenous hemH gene is functionally inactivated; and / or(ii) the endogenous hemQ gene is functionally inactivated.57. The genetically engineered microorganism of any one of paragraphs 49 to 56, which has increased production of succinyl-CoA compared to a corresponding wild-type microorganism.58. The genetically engineered microorganism of any one of paragraphs 49 to 57, which has increased constitutive carbon flux through the glyoxylate cycle compared to a corresponding wild-type microorganism.59. The genetically engineered microorganism of any one of paragraphs 49 to 58, which has increased constitutive expression of the aceBAK operon compared to a corresponding wild-type microorganism.60. The genetically engineered microorganism of any one of paragraphs 49 to 59, which has increased expression of an enzyme of the glyoxylate cycle, increased expression of an activator of the glyoxylate cycle, and / or decreased expression or functional inactivation of a repressor of the glyoxylate cycle.61. The genetically engineered microorganism of paragraph 60, wherein:(i) the enzyme of the glyoxylate cycle is a malate synthase (e.g. AceB) and / or an isocitrate lyase (e.g. AceA); and / or(ii) the activator of the glyoxylate cycle is FruR and / or IHF; and / or(iii) the repressor of the glyoxylate cycle is IclR and / or ArcA; and / or(iv) iclR and / or arcA has a reduced expression or is functionally inactivated; and / or(v) sdhA, sdhB, sdhC, and / or sdhD has a reduced expression or is functionally inactivated; and / or(vi) IdhA has a reduced expression or is functionally inactivated.62. The genetically engineered microorganism of any one of paragraphs 48 to 61, wherein the bacterium is from the genera Alcaligenes, Arthrobacter , Anaerobiospirillum, Bacillus, Brevibacterium, Corynebacterium, Enterococcus, Erwinia, Escherichia, Gluconob acter, Lactobacillus, Mannheimia, Paenibacillus, Rhizobium, Rhodobacter, Rhodococcus, Staphylococcus, Streptomyces, Synechocystis, and Zymomonas.63. The genetically engineered microorganism of any one of paragraphs 48 to 62, wherein the bacterium is Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum.64. The genetically engineered microorganism of paragraph 48, wherein the bacterium is E. coli, wherein the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE, and wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemC.65. The genetically engineered microorganism of paragraph 64, wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemD.66. The genetically engineered microorganism of paragraph 64 or 65, wherein iclR and sdhA are functionally inactivated.67. The genetically engineered microorganism of any one of paragraphs 64 to 66, wherein IdhA is functionally inactivated.68. The genetically engineered microorganism of paragraph 47, that is a genetically engineered yeast.69. The genetically engineered microorganism of paragraph 68, wherein the yeast is from the genera Aspergillus, Candida, Fusarium, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Toruplosis.70. The genetically engineered microorganism of paragraph 69, wherein the yeast is Saccharomyces cerevisiae o Pichia pastoris.71. The genetically engineered microorganism of any one of paragraphs 49 to 70, wherein the HemA comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:1.72. The genetically engineered microorganism of any one of paragraphs 49 to 71, wherein the HemB comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:2.73. The genetically engineered microorganism of any one of paragraphs 49 to 72, wherein the HemC comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 3 or SEQ ID NO:4.74. The genetically engineered microorganism of any one of paragraphs 49 to 73, wherein the HemD comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:5.75. The genetically engineered microorganism of any one of paragraphs 49 to 74, wherein the HemE comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:6.76. The genetically engineered microorganism of any one of paragraphs 49 to 75, wherein the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.77. The genetically engineered microorganism of any one of paragraphs 47 to 76, which is capable of secreting at least 1 mg / L, at least 2 mg / L, at least 3 mg / L, at least 4 mg / L, or at least 5 mg / L of uroporphyrin III, uroporphyrin I, coproporphyrin III and / or coproheme III after 120 hours of cell culture.

[0119] The invention will now be described by way of non-limiting examples.

[0120] EXAMPLE 1

[0121] Bacterial strains and plasmids

[0122] The parental strain of all engineered strains was E. coli BW25113 (F-, A(araD- araB)567, AlacZ4787(::rmB-3), X-, rph-1, A(rhaD-rhaB)568, hsdR514). E. coli Hl-Control 10G (Lucigen, Middleton, WI, USA) was used for molecular cloning.

[0123] The E. coli strain CPC-Sbm was generated by activating the genomic Sbm operon in BW25113 (i.e., by replacing the 204-bp upstream of the Sbm operon with the FRT-Pfrc cassette) and inactivating the IdhA gene as previously described (Srirangan et al., 2014). To fuse the strongpromoter (Pfrc) with the Sbm operon in the E. coli genome, a modified Z Red-mediated recombination protocol was used.

[0124] Knockouts of sdhA, and iclR, were introduced into CPC-Sbm by Pl phage transduction (Miller, 1992) using the appropriate Keio Collection strains (Coli Genetic Stock Center, Yale University, New Haven, CT) as donors (Baba et al., 2006). To eliminate the cotransduced FRT-KnR-FRT cassette, the transductants were transformed with pCP20 (Cherepanov and Wackemagel, 1995), a temperature-sensitive plasmid expressing a flippase (Flp) recombinase. Plasmid pCP20 was then cured by growing cells at 42°C. The genotypes of derived knockout strains were confirmed by colony polymerase chain reaction using the appropriate verification primer sets.

[0125] Genomic DNA from bacterial cells was extracted with the Qiagen Blood & Tissue DNA Isolation Kit (Hilden, Germany). The source gene was amplified by polymerase chain reaction (PCR) using an appropriate primer set and the genomic DNA of the source organism as the template. Taq DNA polymerase was obtained from New England Biolabs (Ipswich, MA, USA). All synthesized oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA, USA). Plasmids were purified using the Qiagen Miniprep kit. DNA sequencing was performed by The Centre for Applied Genomics (Toronto, ON, Canada).

[0126] Heterologous expression of various heme pathway biosynthetic genes was conducted by creating either one or two expression operons using pK184 (GenBank access #: U00800) as the cloning vector. Heterologous expression of genes / operons cloned in the pK184 vector were regulated under the control of the Ptrcand / or Pgracmax promoter with a strong ribosomal binding site.

[0127] Plasmid pK-hemABCD was constructed first to serve as the template for the other plasmids. The backbone, consisting of p!5A ori and Ptrcwith a kanamycin resistance marker, was amplified from a lab-made plasmid. The hemA gene was amplified by polymerase chain reaction (PCR) from the genomic DNA of R. sphaeroides DSM 158. The hemB, hemC, and hemD genes were amplified from the genomic DNA of E. coli MG1655. Subsequently, these five fragments were Gibson-assembled to form pK-hemABCD.

[0128] To construct pK-hemA, primers were first phosphorylated and then used to amplify a single fragment from pK-hemABCD as template. The resulting fragment was self-ligated to form pK-hemA.

[0129] Similarly, a single fragment was amplified from pK-hemABCD with phosphorylated primers. This fragment was self-ligated to form pK-hemAB. For effectivecoexpression, hemA and hemB were aligned to form an operon hemAB regulated by a common strong trc promoter with an individual strong ribosomal binding site (RBS) for each gene.

[0130] pK-hemABC was constructed using primer sets to amplify the backbone and hemA in the first fragment, and hemB and hemC in the second fragment. These two fragments were Gibson assembled to form pK-hemABC. For effective coexpression, hemA, hemB and hemC were aligned to form an operon hemABC regulated by a common strong trc promoter with an individual strong ribosomal binding RBS for each gene.

[0131] pK-hemABCBwas constructed by first amplifying hemA and hemB as a single fragment from pK-hemABCD. hemC was amplified from the genomic DNA of Bacillus subtilis 168. The backbone was amplified from pK-hemABCD. These three fragments were Gibson assembled to form pK-hemABCB. For effective coexpression, hemA, hemB and hemCBwere aligned to form an operon hemABCBregulated by a common strong trc promoter with an individual strong RBS for each gene.

[0132] pK-hemABD was constructed by amplifying hemA and hemB, and amplifying hemD along with the backbone, using pK-hemABCD as template. These two fragments were Gibson assembled to form pK-hemABD. For effective coexpression, hemA, hemB and hemD were aligned to form an operon hemABD regulated by a common strong trc promoter with an individual strong RBS for each gene.

[0133] Plasmid pK-hemABCD-E was constructed first to serve as the template for the other plasmids containing two operons. pK-hemABCD-E was constructed by amplifying hemA along with the backbone (consisting of pl 5 A ori and Ptrcwith a kanamycin resistance marker) and amplifying hemB, hemC and hemD using pK-hemABCD as template. The hemE gene was amplified from the genomic DNA of E. coli MG1655. The Pgracmax WHS amplified from a lab-made plasmid. Subsequently, these five fragments were Gibson-assembled to form pK-hemABCD-E. For effective coexpression, hemA, hemB, hemC and hemD were aligned to form an operon hemABCD regulated by a common strong trc promoter and hemE was included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0134] pK-hemABD-E was constructed by amplifying hemA and hemB, and amplifying hemD and hemE along with the backbone, using pK-hemABCD-E as template. These two fragments were Gibson assembled to form pK-hemABD-E. For effective coexpression, hemA, hemB and hemD were aligned to form an operon hemABD regulated by a common strong trc promoter and hemD was included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0135] pK-hemAB-DE was constructed by amplifying hemA and hemB using pK- hemABCD as template. Pgracmax, and hemD were amplified from pK-hemABD-E separately. hemE was amplified along with the backbone, using pK-hemABD-E as template. These four fragments were Gibson assembled to form pK-hemABD-E. hemA and hemB were aligned to form an operon hemAB regulated by a common strong trc promoter and hemD and hemE were included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0136] pK-hemAB-E was constructed by amplifying hemA and hemB, and amplifying Pgmcmax and hemE along with the backbone, using pK-hemABCD-E as template. These two fragments were Gibson assembled to form pK-hemAB-E. hemA and hemB were aligned to form an operon hemAB regulated by a common strong trc promoter and hemE was included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0137] pK-hemABD-EHQ was constructed by amplifying hemA along with the backbone, and amplifying hemB, hemD and hemE, using pK-hemABD-E as template. The hemH and hemQ genes were amplified from the genomic DNA of B. Subtilis strain 168. These four fragments were Gibson assembled to form pK-hemABD-EHQ. For effective coexpression, hemA, hemB and hemD were aligned to form an operon hemABD regulated by a common strong trc promoter and hemEHQ were included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0138] pK-hemAB-EHQ was constructed by amplifying hemA along with the backbone, and amplifying hemB and hemE, using pK-hemAB-E as template. The hemH and hemQ genes were amplified from the genomic DNA of B. Subtilis strain 168. These four fragments were Gibson assembled to form pK-hemAB-EHQ. For effective coexpression, hemA and hemB were aligned to form an operon hemAB regulated by a common strong trc promoter and hemEHQ were included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0139] pK-hemABD-EH was constructed by amplifying hemA along with the backbone, and amplifying hemB, hemD, hemE and hemH, using pK-hemABD-EHQ as template. These two fragments were Gibson assembled to form pK-hemABD-EH. For effective coexpression, hemA, hemB and hemD were aligned to form an operon hemABD regulated by a common strong trc promoter and hemEH were included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0140] pK-hemAB-EH was constructed by amplifying hemA, hemB and hemE, and amplifying the backbone, using pK-hemAB-E as template. The hemH gene was amplified fromthe genomic DNA of B. Subtilis strain 168. These three fragments were Gibson assembled to form pK-hemAB-EH. For effective coexpression, hemA and hemB were aligned to form an operon hemAB regulated by a common strong trc promoter and hemEH were included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0141] pK-hemABE-H was constructed by amplifying hemA and hemB, and amplifying hemE and PgraCmax separately, using pK-hemABCD-E as template. The hemH was amplified along with the backbone, using pK-hemAB-EH as template. These four fragments were Gibson assembled to form pK-hemABE-H. hemA, hemB and hemE were aligned to form an operon hemABE regulated by a common strong trc promoter and hemH was included on the second operon regulated by the gracmax promoter with an individual strong RBS for each gene.

[0142] Competent cells and transformation

[0143] To prepare chemically competent cells, bacterial cells on the overnight plate were used to inoculate the prewarmed Luria-Bertani (LB) medium with the initial optical density at 600 nm (OD600) of -0.1. Cells were harvested at OD600 of ~0.5 and then washed with ice-cold 15% glycerol three times. Finally, cells were concentrated 230 times and 40 pl of the competent cells were used per transformation. Approximately 100 ng of the plasmid were mixed with 40 pl of electrocompetent cells. Mixture of the cells and DNA were electroporated and recovered in 1 mL SOC medium for 1.5 hours and then spread on selective LB agar plates and incubated at 37 °C overnight.

[0144] Media and cell cultivation

[0145] All medium components were obtained from Sigma-Aldrich Co. (St Louis, MO, USA) except yeast extract and tryptone which were obtained from BD Diagnostic Systems (Franklin Lakes, NJ, USA). E. coli strains, stored as glycerol stocks at -80°C, were streaked on lysogeny broth (LB; 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl) agar plates with appropriate antibiotics (for example, ampicillin (100 mg / L), kanamycin (50 mg / L), and chloramphenicol (40 mg / L)) and incubated at 37°C for 14-16 h.

[0146] For bioreactor cultivation, individual single colonies were picked from LB plates to inoculate 12 mL of super broth (SB) medium (32 g / L tryptone, 20 g / L yeast extract, and 5 g / L NaCl) in a 125 mL conical flask. The culture was incubated at 37 °C and 280 revolutions per min (rpm) using a rotary shaker (New Brunswick Scientific) for 4-6 h and subsequently used as a seed culture to inoculate 220 mL of SB medium at a 2% (vol / vol) concentration in a 1 L conical flask. This second seed culture was incubated at 37 °C and 280 rpm for 14-16 h. Cells were harvested by centrifugation at 4,500 g and 20 °C for 8 min and resuspended in 30 mL of fresh SB medium.The resuspended culture was used to inoculate a stirred tank bioreactor (CelliGen 115, Eppendorf AG) with a working volume of 0.8 L at 37 °C and 430 rpm. The semi-defined production medium in the batch bioreactor contained 30 g / L glycerol, 0.23 g / L K2HPO4, 0.51 g / L NH4CI, 49.8 mg / L MgCh, 48.1 mg / L K2SO4, 1.52 mg / L FeSCU, 0.055 mg / L CaCl2, 2.93 g / L NaCl, 0.72 g / L tricine, 10 g / L yeast extract, 10 mM NaHCCL, and 1 mL / L trace elements (2.86 g / L H3BO3, 1.81 g / L MnCh 4H2O, 0.222 g / L ZnSO47H2O, 0.39 g / L Na2MoO42H2O, 79 pg / L CuSO45H2O, 49.4 pg / L Co(NOs)2 6H2O); (Neidhardt, Bloch, & Smith, 1974), supplemented with 0.05 mM isopropyl P-D-l -thiogalactopyranoside (IPTG). To avoid glycine limiting during the cultivation, 2 g of glycine was supplemented into the bioreactor ~30 h after the inoculation. The pH of the bioreactor culture was maintained at 7.0 ± 0.1 using 3 M NH4OH and 3 M H3PO4.

[0147] Aerobic condition in the bioreactor was maintained by continuously purging the air into the bulk culture at 1 volume of air per volume of liquid per min (vvm).

[0148] Cell density and metabolite quantification

[0149] Culture samples were diluted with 0.15 M saline solution for measuring cell density. All culture samples were washed one time with 0.15 M saline solution prior to ODeoo measurement using a spectrophotometer (DU520, Beckman Coulter, Fullerton, CA). Cell-free medium was prepared by centrifugation of the culture sample at 17.000 / g for 1 minute and filter sterilization using a 0.2 pM syringe filter.

[0150] The quantification of extracellular metabolites and glycerol was conducted using high-performance liquid chromatography (HPLC) (LC-10AT, Shimadzu, Kyoto, Japan) with a refractive index detector (RID; RID-10A, Shimadzu, Kyoto, Japan) and a chromatographic column (Aminex HPX-87H, Bio-Rad Laboratories, CA, USA). The HPLC column temperature was maintained at 35°C and the mobile phase was 5 mM H2SO4 (pH 2) running at 0.6 mL / min. The RID signal was acquired and processed by a data processing unit (Clarity Lite, DataApex, Prague, Czech Republic).

[0151] The concentrations of 5 -ALA and PBG in the cell-free medium were determined using an assay with a modified Ehrlich's reagent (Mauzerall & Granick, 1956).

[0152] To determine intracellular metabolites levels, approximately 2 mL E. coli culture was pelleted by centrifuging twice at 10.000 / g for 2 minutes before resuspension in 2 mL of 50 mM TrisHCl buffer (pH 7.2) and stirring for 20 minutes at room temperature. The resuspended cells were sonicated for 5 minutes and centrifuged at 13.000 / g for 8 minutes before the addition of 2 mL of acetonitrile: 1.7 M HC1 (8:2, v / v) to the pellet, which was then placed in a shaker for 20 minutes. The mixture was separated into a two-phase liquid-liquid system via addition of 0.5mL of saturated MgSO4(aq) and 50 mg of NaCl(s) and then centrifuged at 13,000*g for 5 minutes. The upper organic layer was filtered and, if necessary, diluted with pure acetonitrile prior to HPLC analysis.

[0153] Porphyrins were analyzed using an HPLC (2690 separation module, Waters™, Milford, USA) with a photodiode array (PDA; 2996 PDA detector, Waters™, Milford, USA) detector and a chromatographic column (Chromolith® HighResolution RP-18 endcapped, Supelco). The UV absorbance was measured at 400 nm and the signal was analyzed using a data processing software (Empower 3, Waters™, Milford, USA). A previously described mobile-phase system was employed (Kwon et al., 2003) with minor modifications. Briefly, the mobile phase consisted of two solvent mixtures: solvent A (7.7 g / L ammonium acetate, 125 mL / L acetonitrile, adjusted to pH 5.17 with glacial acetic acid) and solvent B (a methanol-glacial acetic acid mixture with a volume ratio at 10: 1) (Kwon et al., 2003). The flow rate of the mobile phase was 1 mL / min. An isocratic elution step was used with 100% solvent A for 5 min, followed by a linear gradient from 0 to 100% solvent B over 30 min, and further followed by 100% solvent B for 5 min. The column temperature was maintained at 45 °C. The chromatographic peak assignment was confirmed using neat standards and the concentration was determined by preparation of standard curves.

[0154] The percentage yield of UP-III and UP -I was calculated as the mole ratio of the produced UP-III or UP -I to the theoretical maximum UP-III or UP-I production based on the consumed glycerol, assuming a one-to-sixteen molar ratio (i.e., one mole UP-III or UP-I produced per sixteen moles of glycerol consumed). Note that eight moles of succinyl-CoA (derived from 16 moles of glycerol) and eight moles of glycine are needed to generate one mole UP-III or UP-I. The same calculation was used for the percentage yield of CP-III, CP-I, Fe-CP-III, Fe-CP-I, PP-IX and Heme.

[0155] EXAMPLE 2

[0156] Heterologous production of UP in engineered A'. coli

[0157] Strain engineering, transformation, cultivation, cell density and metabolite quantification are as described in Example 1.

[0158] Heterologous production of UP in engineered E. coli expressing HemA

[0159] The Shemin / C4 pathway was implemented by heterologous expression of the plasmid pK-hemA in E. coli CPCS\)mAiclRAsdhA. The double mutation of AiclRAsdhA ensured an abundant supply of precursors as the dissimilated carbon flux in the TCA cycle was directed towards succinyl-CoA under aerobic conditions. This strain generated peak levels of 5-ALA andPBG of 215 mg / L (22.6 mg / ODeoo / L) and 150 mg / L (15.9 mg / ODeoo / L), respectively at 71 h (Figure 3A). The final extracellular titers of UP-I and UP -III were 55.3 mg / L (0.32% yield) and 41.9 mg / L (0.25% yield), respectively (Figure 3B). Significant pigmentation was observed in the cell free medium (CFM) and cell pellet samples. A deep reddish hue in the CFM and cell pellet samples were visible after 30 hours of cultivation. After 71 hours and 96 hours, respectively, the CFM and cell pellet samples were a dark red-brown.

[0160] Heterologous production of UP in engineered E. coli expressing HemA and HemB

[0161] The plasmid pK-hemAB was expressed in E. coli CPCSbmAiclRAsdhA. This strain of E. coli produced peak 5-ALA and PBG levels of 304.3 mg / L (20.9 mg / OD600 / L) and 1995 mg / L (193 mg / OD600 / L), respectively, at 48 h (Figure 4A). The final extracellular titers of UP-I and UP -III were 555.3 mg / L (3.7% yield) and 346.6 mg / L (2.3% yield), respectively (Figure 4B). Significant pigmentation was observed in the CFM and the cell pellet samples. A deep reddish hue in the CFM and cell pellet samples were visible after 25 hours of cultivation. After 47 hours and 121 hours, respectively, the CFM and cell pellet samples were a dark red-brown.

[0162] Heterologous production of UP in engineered E. coli expressing HemA, HemB, and HemC

[0163] The expression of plasmid pK-hemABC, containing E. coli hemC, in E. coli CPC- SbmAiclBAsdhA surprisingly resulted in a cell culture that exhibited no pigmentation. This strain produced low levels of 5-ALA and PBG, only up to 14 mg / L (0.84 mg / ODeoo / L) and 11 mg / L (0.67 mg / ODeoo / L), respectively (Figure 5), which were similar to those of the control strain (CPC- SbmAiclRAsdhA). Without being bound by theory, these findings suggest that the overexpression of the native E. coli hemC gene potentially disrupted the metabolic activity of the Shemin / C4 pathway.

[0164] By contrast, the expression of plasmid pK-hemABCB, containing B. subtilis hemC, in E. coli CPC- bmAiclRAsdhA resulted in a cell culture that displayed significant pigmentation by day 2. Furthermore, E. coli overexpressing hemC from B. subtilis exhibited increased production of 5-ALA and PBG in comparison to E. coli overexpressing native hemC, with peak levels being 81.2 mg / L (5.25 mg / OD600 / L) and 108.3 mg / L (7 mg / OD600 / L), respectively (Figure 6A compared to Figure 5). However, significant PBG accumulation was not observed, suggesting that, without being bound by theory, the expressed HemC from B. subtilis effectively converted PBG to HMB in the early cultivation stage (Figure 6A). Nevertheless, E. coli overexpressing hemC from B. subtilis produced significantly lower amounts of UP-I and UP -III compared to E. colioverexpressing hemA and hemB (but not hemC), with peak levels of 200.4 mg / L (1.18% yield) and 135 mg / L (0.8% yield), respectively (Figure 6B).

[0165] Heterologous production of UP in engineered E. coli expressing HemA, HemB,HemD

[0166] The plasmid pK-hemABD was expressed in E. coli CPCS\)mAiclRAsdhA. This strain produced high levels of 5-ALA and PBG, peaking at 196 mg / L (18.2 mg / OD600 / L) and 1117 mg / L (83.16 mg / OD600 / L) at 57 h, respectively (Figure 7A). The final extracellular titers of UP-I and UP-III were 538.9 mg / L and 287.5 mg / L, at 3.18% and 1.7% yields, respectively. (Figure 7B). The distribution of UP-I and UP-III in E. coli which overexpressed hemA (R. sphaeroides), hemB and hemD resembled that of E. coli which overexpressed hemA (R. sphaeroides) and hemB only, suggesting, without being bound by theory, that hemD overexpression did not enhance the metabolic flux toward heme biosynthesis, rather the conversion of HMB via the autoxi dati on reaction was more effective than the enzymatic reaction by HemD even upon hemD overexpression. Significant pigmentation was observed in the CFM and cell pellet samples. A deep reddish hue in the CFM and cell pellet samples were visible after 24 hours of cultivation. After 47 hours and 121 hours, respectively, the CFM and cell pellet samples were a dark red-brown.

[0167] EXAMPLE 3

[0168] Heterologous production of CP in engineered E. coli

[0169] Strain engineering, transformation, cultivation, cell density and metabolite quantification are as described in Example 1.

[0170] Heterologous production of CP in engineered E. coli expressing HemA, HemB, HemD and HemE

[0171] Two different plasmid constructs were used to express HemA, HemB, HemD and HemE in E. coli'. 1) pK-hemABD-E wherein hemABD formed an operon regulated by the trc promoter, and hemE was regulated by the gracmax promoter; or 2) pK-hemAB-DE, wherein hemAB formed an operon regulated by the trc promoter and hemDE formed an operon regulated by the gracmax promoter.

[0172] Cultivation of E. coli CPC-SbmAiclRAsdhA containing pK-hemABD-E produced 5-ALA and PBG, peaking at 99.7 mg / L (5.4 mg / OD600 / L) and 665.7 mg / L (35.7 mg / OD600 / L) at 48 h, respectively (Figure 8 A). The final extracellular titers were 182.1 mg / L and 84.3 mg / L of CP-III and CP-I, at 1.4% and 0.6% yields, respectively and also 20.1 mg / L and 17.5 mg / L of UP- III and UP-I, at 0.12% and 0.1% yields, respectively (Figure 8B). Pigmentation was observed inthe CFM and cell pellet samples. A dark red-brown was visible in the CFM and cell pellet samples, after 96 h and 120 h of cultivation, respectively.

[0173] Cultivation of E. coli CPC-SbmAiclRAsdhA containing pK-hemAB-DE produced 5-ALA and PBG, peaking at 93 mg / L (10 mg / OD600 / L) and 866.9 mg / L (92.7 mg / OD600 / L) respectively, at 48 h (Figure 9A). The final extracellular titers were 119.1 mg / L and 68.2 mg / L of CP-III and CP-I, at 0.89% and 0.51% yields, respectively and also 20.3 mg / L and 18.6 mg / L of UP-III and UP -I, at 0.12% and 0.11% yields, respectively (Figure 9B). Pigmentation was observed in the CFM and cell pellet samples. A reddish-brown color was visible in the CFM and cell pellet after 48 hours. A dark red-brown was visible in the CFM and cell pellet samples, after 120 h of cultivation.

[0174] Heterologous production of CP in engineered E. coli expressing HemA, HemB, andHemE

[0175] Cultivation of E. coli CPC-SbmAiclRAsdhA containing pK-hemAB-E produced 5- ALA and PBG, peaking at 106.7 mg / L (13.33 mg / OD600 / L) and 1180.6 mg / L (147.5 mg / OD600 / L) at 48 h, respectively (Figure 10A). The final extracellular titers were 151 mg / L and 96.3 mg / L of CP-III and CP-I, at 1.13% and 0.72% yields, respectively and also 20.9 mg / L and 20.9 mg / L of UP-III and UP-I, at 0.12% and 0.12% yields, respectively (Figure 10B). The CFM had a deep reddish hue after 48 hours of cultivation. By 120 hours of cultivation, the CFM and cell pellets were both a dark red-brown.

[0176] EXAMPLE 4

[0177] Heterologous production of coproheme (Fe-CP) via the CPD branch in engineered E. coli

[0178] Strain engineering, transformation, cultivation, cell density and metabolite quantification are as described in Example 1.

[0179] Heterologous production of Fe-CP in engineered / / . coli expressing HemA, HemB, HemD. HemE and HemH

[0180] The plasmid pK-hemABD-EH, containing B. subtilis hemH, was expressed in E. coli CPC-SbmAiclRAsdhA. This strain produced 5-ALA and PBG, peaking at 75.5 mg / L (5.3 mg / OD600 / L) and 242.4 mg / L (17.1 mg / OD600 / L) at 48 h, respectively (Figure 11 A). The final extracellular titers were 18.8 mg / L and 15.4 mg / L of Fe-CP-III and Fe-CP-I, at 0.13% and 0.11% yields, respectively and also 10.6 mg / L and 1.2 mg / L of CP-III and CP-I, at 0.08% and 0.01% yields, respectively (Figure 11B). The final intracellular titers of Fe-CP-III was 5.1 mg / L, at a 0.04% yield (Figure 1 IB). CFM and cell pellet samples showed a deep reddish hue after 48 hoursof cultivation. After 120 hours of cultivation, the CFM and cell pellet samples were a dark red- brown.

[0181] Heterologous production of Fe-CP in engineered / / . coli expressing HemA, HemB,HemE and HemH

[0182] Two different plasmid constructs were used to express HemA, HemB, HemE and HemH in E. coli'. 1) pK-hemAB-EH wherein hemAB formed an operon regulated by the trc promoter, and hemEH formed an operon regulated by the gracmax promoter; or 2) pK-hemABE- H, wherein hemABE formed an operon regulated by the trc promoter and hemH was regulated by the gracmax promoter. Both constructs contained B. subtilis hemH.

[0183] Cultivation of E. coli CPC-SbmAiclRAsdhA containing pK-hemAB-EH produced 5-ALA and PBG, peaking at 260.7 mg / L (14.1 mg / OD600 / L) and 926.2 mg / L (50.1 mg / OD600 / L) at 48 h, respectively (Figure 12A). The final extracellular titers were 34.3 mg / L and 26.9 mg / L of Fe-CP-III and Fe-CP-I, at 0.24% and 0.19% yields, respectively and also 13.8 mg / L and 3.4 mg / L of CP-III and CP-I, at 0.1% and 0.03% yields, respectively (Figure 12B). The final intracellular titers of Fe-CP-III, Fe-CP-I and CP-III were 7.9 mg / L, 16 mg / L and 0.5 mg / L at 0.05%, 0.11% and 0.004% yields, respectively (Figure 12B). CFM and cell pellet samples showed a deep reddish hue after 48 hours of cultivation. After 120 hours of cultivation, the CFM and cell pellet samples were dark red-brown.

[0184] Cultivation of E. coli CPC-SbmAiclRAsdhA containing pK-hemABE-H produced 5-ALA and PBG, peaking at 155.7 mg / L (10.9 mg / OD600 / L) and 918.4 mg / L (64.2 mg / OD600 / L) at 48 h, respectively (Figure 13 A). The final extracellular titers were 42.5 mg / L and 44.3 mg / L of Fe-CP-III and Fe-CP-I, at 0.29% and 0.31% yields, respectively and also 17.5 mg / L and 1.5 mg / L of CP-III and CP-I, at 0.13% and 0.01% yields, respectively (Figure 13B). The final intracellular titers of Fe-CP-III and Fe-CP-I and CP-III were 2.5 mg / L, 2.5 mg / L and 0 mg / L at 0.02%, 0.02% and 0% yields, respectively (Figure 13B). Pigmentation was observed in the CFM and cell pellet samples. A slight reddish-brown color was visible in the CFM and cell pellet after 24 hours. A dark red-brown was visible in the CFM and cell pellet samples after 120 h of cultivation.

[0185] EXAMPLE 5

[0186] Heterologous production of heme via the CPD branch in engineered E. coli

[0187] Strain engineering, transformation, cultivation, cell density and metabolite quantification are as described in Example 1.

[0188] Heterologous production of heme in engineered E. coli expressing HemA, HemB, HemD. HemE HemH and HemQ

[0189] The plasmid pK-hemABD-EHQ, containing B. subtilis hemH and hemQ, was expressed in / / . coli CPC-SbmAiclRAsdhA. This strain produced 5-ALA and PBG, peaking at 94.7 mg / L (7.2 mg / OD600 / L) and 232.3 mg / L (17.6 mg / OD600 / L) at 48 h, respectively (Figure 14A). The final extracellular titers were 16.7 mg / L, 6.6 mg / L, 2.1 mg / L, 4.5 mg / L and 4.5 mg / L of Fe- CP-III, CP-III, CP-I, UP-III and UP-I, at 0.12%, 0.05%, 0.02%, 0.03%, and 0.03% yields, respectively (Figure 14B). The final intracellular titers of Heme, PP-IX, and Fe-CP-III were 15.8 mg / L, 7.6 mg / L and 3.8 mg / L at 0.13%, 0.06% and 0.02% yields, respectively (Figure 14B). CFM and cell pellet samples showed a deep reddish hue after 48 hours of cultivation. After 120 hours of cultivation, the CFM and cell pellet samples were dark red-brown.

[0190] This strain demonstrates that heme can be produced through the CPD pathway, as opposed to the PPD pathway, by introducing HemH and HemQ from gram positive bacteria (ex. B. subtilis . The detection of a significant concentration of Fe-CP-III (20.5mg / L intra + extracellular) when B. subtilis HemH and HemQ were expressed was evidence of successful implementation of CPD in E. coli. However, without being bound by theory, the biosynthesis of heme in this strain appeared to be limited by intracellular accumulation of heme and PP-IX, both of which have low water solubility. Furthermore, back intracellular accumulation of Fe-CP-III and even CP-III was apparent (Figure 14B).

[0191] Heterologous production of heme in engineered E. coli expressing HemA, HemB, HemE HemH and HemQ

[0192] The plasmid pK-hemAB-EHQ, containing B. subtilis hemH and hemQ, was expressed in E. coli CPC-SbmAiclRAsdhA. This strain produced 5-ALA and PBG, peaking at 143.6 mg / L (6.7 mg / OD600 / L) and 361 mg / L (16.8 mg / OD600 / L) at 48 h, respectively (Figure 15A). The final extracellular titers were 0.2 mg / L, 7.1 mg / L, 26.1 mg / L, 2.2 mg / L and 1 mg / L of Heme, Fe-CP-III, Fe-CP-I, CP-III, and CP-I, at 0.002%, 0.05%, 0.18%, 0.02%, and 0.01% yields, respectively (Figure 15B). The final intracellular titers of Heme, PP-IX, Fe-CP-III and CP-III were 17.8 mg / L, 10.1 mg / L, 7 mg / L and 1.9 mg / L at 0.14%, 0.09%, 0.05% and 0.01% yields, respectively (Figure 15B). CFM and cell pellet samples showed a deep reddish hue after 48 hours of cultivation. After 120 hours of cultivation, the CFM and cell pellet samples were a dark red- brown.

[0193] Notably, the expression of HemD does not appear to be critical as this strain produced similar amounts of heme when compared to the E. coli strain expressing pK-hemABD- EHQ. Without being bound by theory, similar to the E. coli strain expressing pK-hemABD-EHQ, the biosynthesis of heme in this strain also appeared to be limited by intracellular accumulation ofheme and PP-IX. Furthermore, back intracellular accumulation of Fe-CP-III and even CP-III was also apparent (Figure 15B).

[0194] EXAMPLE 6

[0195] Comparing the heterologous production of heme in engineered E. coli via the PPP and the CPD branch

[0196] Strain engineering, transformation, cultivation, cell density and metabolite quantification are as described in Example 1.

[0197] Figure 16A shows the distribution of heme biosynthetic pathway products using the PPD pathway, after cultivation for 120 hours in a bioreactor for E. coli CPC-SbmAiclRAsdhA expressing pK-HemABD-EFGH. This strain predominantly generated intracellular heme at a concentration of approximately 42 mg / L in total. Furthermore, roughly 80% of heme is in the cells.

[0198] By contrast, Figure 16B shows the distribution of heme biosynthetic pathway products using the CPD pathway, after cultivation for 120 hours in a bioreactor for E. coli CPC- SbmAiclRAsdhA expressing pK-HemABD-EHQ as detailed in Example 5, where hemH and hemQ are from B subtilis.

[0199] Figure 17 shows a comparison of the heme biosynthetic pathway product yields from E. coli CPC-SbmAiclRAsdhA containing either pK-HemABD-E (see Example 3), pK- HemAB-E (see Example 3), pK-HemABD-EFGH or pK-HemABD-EHQ (see Example 5). E. coli CPC-SbmAiclRAsdhA expressing either pK-HemABD-E or pK-HemAB-E showed a much higher yield of CP-III than intracellular heme obtained by expressing pK-HemABD-EFGH or pK- HemABD-EHQ. As detailed in Example 7 below, the secreted CP-III can then be converted to heme in vitro, allowing for the production of more heme than if the heme was solely generated intracellularly via the CPD pathway (as in pK-HemABD-EHQ).

[0200] EXAMPLE 7

[0201] Two-step method of heme production

[0202] Preparation of purified HemH and HemQ

[0203] pTrc-His-hemQB. s and pTrc-hemQB. s-His were constructed by amplifying hemQ from B. Subtilis strain 168. Primers were designed to incorporate a 6x His-tag on either the C- terminus or N-terminus. The fragment was then Gibson assembled with the backbone amplified from pTrc99a plasmid. A similar approach was applied for construction of pTrc-His-hemHB. s, pTrc-hemHB. s-His, pTrc-His-hemQs. a and pTrc-hemQs. a-His. pTrc-His-hemQs. a and pTrc- hemQs. a-His contain hemQ from Staphylococcus aureus.

[0204] To purify the HemH and HemQ enzymes, IMAC columns were used. Initially, strain containing the His-tagged protein was used to inoculate 200 mL of the Luria-Bertani (LB) medium with the initial optical density at 600 nm (OD600) of ~0.1. The culture was induced with Isopropyl P-D-l -thiogalactopyranoside (IPTG) at OD600 of ~1. Cells were harvested at 5 hours post-induction at 4 °C. The cell pellet was resuspended in lysis buffer and sonicated for 4 minutes. After centrifugation for 20 minutes at 12000 X g and 4 °C, the supernatant was filtered and applied to an equilibrated 1 mL IMAC cartridge. Subsequently, the column was washed with a low concentration imidazole wash buffer and finally, the enzyme was eluted with an imidazole-rich elution buffer. The purified enzyme was concentrated using Amicon Centrifugal filters and the concentration was determined using the Bradford assay.

[0205] Two-step method of heme production

[0206] This example demonstrates the in vitro conversion of Fe-CP-III into heme using cell lysate containing HemQ. E. coli strain CPC-SbmAiclRAsdhA expressing pK-hemAB-EH was cultivated in a fermenter for 144 hours. The strain was engineered and cultivated as described in Example 1. The final intracellular and extracellular titers of heme biosynthetic products were measured by HPLC as described in Example 1. The final amount of extracellular Fe-CP-III was 34.3 mg / L (Figure 18A). CFM containing 32 mg / L of Fe-CP-III was treated wiX Bacillus subtilis HemQ-his rich lysate for 1 hour (Figure 18B) or overnight (Figure 18C). Thirty -two mg / L of Fe- CP-III was converted into 12 mg / L of heme. Alternatively, the CFM containing 32 mg / L of Fe- CP-III was treated with purified Bacillus subtilis HemQ-his overnight (Figure 19) Twenty-five mg / L of Fe-CP-III was converted to 8 mg / L of heme.

[0207] The in vitro conversion of Fe-CP-III into heme can also be conducted using Staphylococcus aureus HemQ-his rich lysate or purified Staphylococcus aureus HemQ. E. coli strain CPC-SbmAiclRAsdhA expressing pK-hemABE-H was cultivated in a fermenter for 144 hours. The strain was engineered and cultivated as described in Example 1. The final intracellular and extracellular titers of heme biosynthetic products were measured by HPLC as described in Example 1. The final amount of extracellular Fe-CP-III was 42.5 mg / L. CFM containing 35 mg / L of Fe-CP-III was treated with purified Bacillus subtilis HemQ-his overnight (Figure 20). Thirty- five mg / L of Fe-CP-III was converted to 12 mg / L of heme.

[0208] It is to be understood that the disclosure is not limited to the disclosed Examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0209] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0210] REFERENCESAltschul, S.F. et al (1990). Basic local alignment search tool. Journal of molecular biology, 215, 403-410.Altschul, S.F. etal (1997). Gapped BLAST and PSI-BLAST: anew generation of protein database search programs. Nucleic acids research, 25, 3389-3402.Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Molecular Systems Biology, 2, 1-11.Cherepanov, P.P. and Wackemagel W. (1995). Gene disruption in Escherichia coli'. TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 158(1), 9-14.Karlin, S. and Altschul, S.F. (1990). Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proceedings of the National Academy of Sciences 87, 2264-2268.Karlin, S. and Altschul, S.F. (1993). Applications and statistics for multiple high-scoring segments in molecular sequences. Proceedings of the National Academy of Sciences 90, 5873-5877.Kwon, S. J., De Boer, A. L., Petri, R., & Schmidt-Dannert, C. (2003). High-Level Production of Porphyrins in Metabolically Engineered Escherichia coli: Systematic Extension of a Pathway Assembled from Overexpressed Genes Involved in Heme Biosynthesis. Applied and Environmental Microbiology, 69( ), 4875-4883. doi: 10.1128 / aem.69.8.4875-4883.2003Mauzerall, D., and Granick, S. (1956). The occurrence and determination of 5-aminolevulinic acid and porphobilinogen in urine. The Journal of biological chemistry, 219(1), 435-446. doi:10.1016 / S0021-9258(18)65809-0Miller, J. H. (1992). A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria (pp. 1-876). New York, NY : Cold Spring Harbor Laboratory Press.Myers, E. and Miller, W. (1988). Optimal alignments in linear space. CABIOS, 4, 11-17.Srirangan, K., Liu, X., Westbrook, A., Akawi, L., Pyne, M.E., Moo-Young, M., and Chou, C.P. (2014). Biochemical, genetic, and metabolic engineering strategies to enhance coproduction of 1- propanol and ethanol in engineered Escherichia coli. Applied Microbiology and Biotechnology, 98(22),9499-9515. doi: 10.1007 / s00253-014-6093-9

Claims

CLAIMS:

1. A method of producing heme, comprising:(a) culturing a microorganism that secretes coproporphyrin III and / or coproheme III in a culture medium; and(b) converting the secreted coproporphyrin III and / or coproheme III into heme.

2. The method of claim 1, wherein the coproporphyrin III and / or coproheme III is converted into heme in the culture medium.

3. The method of claim 2, wherein the culture medium comprises HemQ and optionally comprises HemH.

4. The method of claim 1, wherein:(i) step (b) comprises adding HemQ to the culture medium and optionally adding HemH to the culture medium; or(ii) step (b) comprises isolating the secreted coproporphyrin III and / or coproheme III and contacting the isolated coproporphyrin III and / or coproheme III with a solution comprising HemQ and optionally comprising HemH.

5. The method of claim 1, wherein step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a second microorganism that secretes HemQ and optionally secreted HemH.

6. The method of claim 5, wherein step (a) further comprises co-culturing the microorganism that secretes coproporphyrin III and / or coproheme III with a third microorganism that secretes HemH.

7. The method of claim 3, wherein:(i) the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; and / or(ii) the HemQ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NOTO or SEQ ID NO: 11.

8. The method of claim 1, wherein the microorganism that secretes coproporphyrin III and / or coproheme III is a genetically engineered microorganism, optionally wherein the genetically engineered microorganism is a bacterium.

9. The method of claim 8, wherein the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, HemD, HemE, HemH, HemQ or combinations thereof.

10. The method of claim 9, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encodingHemA; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemB; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemC; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemD; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemC, and HemD.

11. The method of claim 9, wherein:(i) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE; or(ii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA and HemE; or(iii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB and HemE; or(iv) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, and HemE; or(v) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, and HemE; or(vi) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemE and HernH; or(vii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemE, and HernH; or(viii) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemB, HemE, and HernH; or(ix) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemE, and HernH; or(x) the bacterium comprises at least one exogenous nucleic acid molecule encoding HemA, HemB, HemD, HemE, and HernH.

12. The method of claim 9, wherein:(i) the bacterium is E. coli, and wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemC; or(ii) the bacterium is E. coli, and wherein the at least one exogenous nucleic acid molecule further encodes the HemC of B. subtilis.

13. The method of claim 9, wherein the bacterium does not comprise an exogenous nucleic acid molecule encoding HemD.

14. The method of claim 11, wherein the bacterium is not E. colt, and wherein the at least one exogenous nucleic acid molecule further encodes HemC.

15. The method of claim 9, wherein:(i) the endogenous hemH gene is functionally inactivated; and / or(ii) the endogenous hemQ gene is functionally inactivated.

16. The method of claim 9, wherein the bacterium has:(i) increased production of succinyl-CoA compared to a corresponding wild-type microorganism; and / or(ii) increased constitutive carbon flux through the glyoxylate cycle compared to a corresponding wild-type microorganism; and / or(iii) increased constitutive expression of the aceBAK operon compared to a corresponding wild-type microorganism.

17. The method of claim 9, wherein the bacterium has increased expression of an enzyme of the glyoxylate cycle, increased expression of an activator of the glyoxylate cycle, and / or decreased expression or functional inactivation of a repressor of the glyoxylate cycle.

18. The method of claim 17, wherein:(i) the enzyme of the glyoxylate cycle is a malate synthase (e.g. AceB) and / or an isocitrate lyase (e.g. AceA); and / or(ii) the activator of the glyoxylate cycle is FruR and / or IHF; and / or(iii) the repressor of the glyoxylate cycle is IclR and / or ArcA; and / or(iv) iclR and / or arcA has a reduced expression or is functionally inactivated; and / or(v) sdhA, sdhB, sdhC, and / or sdhD has a reduced expression or is functionally inactivated; and / or(vi) IdhA has a reduced expression or is functionally inactivated.

19. The method of claim 9, wherein:(i) the HemA comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:1; and / or(ii) the HemB comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:2; and / or(iii) the HemC comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:3 or SEQ ID NO:4; and / or(iv) the HemD comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:5; and / or(v) the HemE comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:6; and / or(vi) the HemH comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

20. A genetically engineered microorganism capable of secreting uroporphyrin III, uroporphyrin I, coproporphyrin III and / or coproheme III.

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