CN111213579A - Method for overproducing hemoglobin in algae and compositions therefrom - Google Patents

Abstract

The present invention provides compositions and methods for producing compositions from algae that overproduce heme. Also provided are methods of growing algae that overproduce hemoglobin, methods of isolating hemoglobin-containing fractions from algae cultures, and compositions and methods of making food products using hemoglobin produced by algae. The present invention provides algal strains and methods of selecting algal strains that overproduce heme. Also provided are compositions, including edible compositions, that contain heme produced by algae.

Description

Method for overproducing hemoglobin in algae and compositions therefrom

Background

With the advent of industrial animal farming, the consumption of animal meat continues to increase. Animal agriculture accounts for more than 18% of the greenhouse gases produced so far, and is one of the main causes of climate change. In addition to land use, animal farming requires large amounts of fresh water, a resource that is increasingly difficult to acquire. It is estimated that 1799 gallons of fresh water is required to produce one pound of beef, and 576 gallons of water is required to produce one pound of pork. This is compared to 216 gallons of fresh water for 1 pound of soybeans or 108 gallons for 1 pound of corn. The strength of the fresh water required to produce animal meat is a result of the inefficiency of the animal to consume the water required for plant growth and the food the animal consumes to convert into actual meat.

To address the sustainability and ethical issues of animal meat consumption, the food industry has been actively attempting to develop plant-based alternatives that have the same taste, feel and odor as meat products. However, many plant-based alternatives have not been able to penetrate the larger food and consumer markets at the present time. Typically, these substitutes consist of plant-based materials that are extruded to produce a firm texture to improve mouthfeel and then mixed with various flavors and aromas to form compounds to improve the taste and odor of these products. Unfortunately, these alternatives are largely attractive to consumers who have been dedicated to a vegetarian/vegetarian lifestyle, rather than those who are accustomed to eating meat. To increase the sustainability of the food ecosystem, products must be developed that appeal to consumers who currently prefer meat. By producing the next generation of plant products, the contribution of greenhouse gases and water demand from animal agriculture can be greatly reduced.

Recent advances have demonstrated the potential of using heme-containing proteins purified from host organisms to bring the taste and aroma characteristics of products closer to those of meat. It is believed that heme from heme-containing proteins is responsible for imparting "meaty" taste and aroma to meat products. However, available sources of heme-containing proteins are expensive and technically focused, limiting their utility. For example, the hemopexin, leghemoglobin, has been extracted from soybean roots, but this approach has proven to be expensive, making it economically less feasible to incorporate it into meat substitutes. The yeast, Pichia pastoris, has been designed to express heme binding proteins, for example with 8 additional enzymatic pathways for the production of heme molecules. This approach still requires purification of the hemopexin from the expression host before its incorporation into the finished product, which limits the potential positive impact due to economic limitations. In addition to being economically inefficient, the product has been genetically modified to make it less attractive to many consumers who choose to consume foods that are not genetically engineered.

Disclosure of Invention

To address the economic and consumer issues associated with current methods of incorporating heme into products, compositions and methods for producing such compositions from algae that overproduce heme are provided herein. Algae with overproduction of heme can be incorporated into finished products without expensive purification methods.

Also provided herein are methods of growing algae with overproduction of heme, methods of isolating heme-containing fractions from algae cultures, and compositions and methods of making food products using heme produced by algae. Provided herein are strains and methods of selecting for heme overproducing strains.

Compositions, including edible compositions containing heme produced by algae, are also provided. In some embodiments, the compositions are suitable as food ingredients and include preparations from algal strains that overexpress (i.e., overproduce) heme. In some embodiments, the formulation is biomass from an algal strain. In some embodiments, the preparation is a stratified (fractionated) biomass from an algal strain. In some embodiments, the layered biomass is a heme-rich component (fraction). In some embodiments, the heme-rich component is also protein-rich. In some embodiments, the preparation from the algal strain that overexpresses heme is an extracellular component of an algal culture. In some formulation embodiments, the formulation is purified from a protein.

In some embodiments, the preparation from the algal strain that overexpresses heme imparts a red color or a color similar to red to the composition. In some embodiments, the formulation imparts a meat or meat-like taste to the composition. In some embodiments, the formulation imparts a meat or meat-like texture to the composition. In some embodiments, the composition is used as an ingredient in a food product. In some embodiments, the food product is clean meat, cultured meat, synthetic meat, plant-based meat, or non-animal cell-based meat.

In some embodiments, the algae used in the formulation is of the species Chlamydomonas (Chlamydomonas).

Also provided herein are methods of producing heme compositions. In some embodiments, the method comprises growing a culture of algae, wherein the algae is a heme overproducer, and isolating a heme composition from the culture. In some embodiments, the algae are grown by aerobic fermentation. In some embodiments, the algae used in the method comprises chloroplasts. In some embodiments, heme biosynthesis occurs in a chloroplast.

In some embodiments of the methods and compositions provided herein, the algae lacks the ability to produce chlorophyll. In some embodiments, the algae lacks the ability to produce functional Mg-chelatases. In some embodiments, the algae lacks a functional ChlD1, ChlD2, or ChlDH gene product. In some embodiments, the algae lacks a functional light-dependent chlorophyllin ester. In some embodiments, the algae lacks functional light-independent ortho-chlorophyllin. In some embodiments, the algae lacks a functional ChlB, ChlL, or ChlN gene product.

In some embodiments of the methods and compositions provided herein, the algae overexpresses a gene selected from the group consisting of: glutamyl-tRNA reductase, glutamyl-1-semialdehyde transaminase, ALA dehydrogenase, porphobilinogen deaminase, UPGIII synthase, UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelatase.

In some embodiments, the algae used in the methods and compositions herein are produced by mating (mating), and wherein the derived strain is red or a color similar to red. In some embodiments, the algae are produced by mutagenesis. In some embodiments, mating or mutagenesis produces red or red-like color algae for use in compositions and methods of producing compositions.

In some embodiments of the methods of producing a heme composition, heme is isolated as part of algal biomass. In some embodiments, heme is contained in the stratified algal biomass. In some embodiments, the layered biomass is also protein-rich. In some embodiments, the heme-containing composition is isolated from an extracellular medium of an algal culture. In some embodiments, the method comprises separating the heme composition from the algal proteins. In some embodiments, the composition is produced by an alga that lacks carotenoids.

In some embodiments of the methods for producing a heme composition, the algae is a species of Chlamydomonas (Chlamydomonas). In some embodiments, the algae is chlamydomonas reinhardtii (Chlamydomonasreinhardtii).

In some embodiments of the methods herein, heme-overexpressing algal strains used in the methods are obtained by mating strains that grow faster than wild-type algae in the dark under the same conditions with strains that already exhibit a red color or a color similar to red. In some embodiments, the heme overproducer algae is a strain produced by mating a carotenoid-deficient strain with a strain exhibiting a red color or a color similar to red. In some embodiments, heme-overproducer algae are produced by mutagenizing a first starting strain and selecting a second strain that grows faster in the dark than the first starting strain. In some embodiments, the heme-overproducer algae are produced by mutagenizing a first starting strain and selecting a second strain that lacks one or more carotenoids.

The methods herein can be used to provide a heme composition for use as a food or food ingredient. In some embodiments, the heme composition imparts a red color or a color similar to a red color to a product or portion thereof. In some embodiments, the heme composition imparts a meat or meat-like taste to the product or portion thereof. In some embodiments, the heme composition imparts a meat or meat-like texture to a product or portion thereof. The methods herein can also be used to provide isolated heme (such as by purification from algae). In some embodiments, the resulting composition comprises the use of an alga lacking carotenoids.

Also provided herein are methods of producing a heme-containing composition, comprising the steps of: (a) culturing a strain of algae under dark conditions, wherein the culturing is performed in the dark, wherein the strain produces no or reduced chlorophyll production, and (b) collecting a portion of the algal culture. In some embodiments of the method, the algae are of the species Chlamydomonas (Chlamydomonas). In some embodiments, the algae is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii). In some embodiments of the method, the algal protoporphyrinogen IX is increased. In some embodiments, the algae exhibits a red color or a color similar to red when grown under dark conditions. In some embodiments, the algal strain lacks one or more functional gene products selected from the group consisting of: ChlD, ChlD2, CH1H, light-activated protochlorophyllin oxidoreductase and light-independent protochlorophyllin gene products.

In some embodiments of the methods for producing a heme-containing composition, the algal strain has increased ferrochelatase expression as compared to a wild-type algal strain. In some embodiments, the algal strain has an increased amount of heme, heme-containing protein, protoporphyrinogen IX, biliverdin IX, phytochrome (phytochromobin), ferrochelatase, or a combination thereof. In some embodiments, the algal strain lacks one or more of a magnesium chelatase, magnesium protoporphyrinogen IX, a protoporphyrinogen ester, a chlorophyllin ester, and chlorophyll.

In some embodiments of the methods for producing a heme-containing composition, the algae are grown by aerobic fermentation. In some embodiments, the algae is grown to a density of greater than about 10g/L, about 20g/L, about 30g/L, about 40g/L, about 50g/L, about 75g/L, about 100g/L, about 125g/L, or about 150 g/liter. In some embodiments, algae are grown using acetate as a source of reducing carbon (reduced carbon source). In some embodiments, the algae are grown using sugars as a reducing carbon source. In some embodiments, the algal culture is supplemented with iron during the culturing step. In some embodiments, the algal culture is inoculated at a density greater than about 0.1g/L, about 1.0g/L, about 5.0g/L, about 10g/L, about 20g/L, about 50g/L, about 80g/L, or about 100 g/L.

In some embodiments, the collected portion of the algal culture exhibits a red color or a color similar to red. In some embodiments, the collected portion is extracellular medium from an algal culture. In some embodiments, the collected portion is biomass from an algal culture or stratified biomass. In some embodiments, layering the collected fraction removes substantially all or most of the carotenoids from the fraction. In some embodiments, layering the collected portion removes substantially all or most of the starch from the portion. In some embodiments, the fractions collected are stratified to produce a protein-rich fraction. In some embodiments, the collected fraction is a heme-rich fraction or purified heme.

In some embodiments of the method, a cleaned meat product is produced, and the method further comprises combining the collected portion with a cleaned meat-making composition, wherein the collected portion provides a red color or a red-like color to the cleaned meat product.

In some embodiments, the methods and compositions provided herein comprise recombinant algae. In other embodiments, the methods and compositions provided herein do not include recombinant algae, and the algal strain is not a transgenic strain.

Drawings

Fig. 1 is a schematic diagram showing an exemplary pathway for the production of heme in algae. This exemplary pathway can be used to produce chlorophyll by wild-type algae, but it can also be used to produce heme.

Fig. 2 is a schematic diagram showing exemplary layering of algae overexpressing heme, showing separation into protein and heme-rich biomass, which is separated from starch and carotenoid components.

FIG. 3 is a graph showing an exemplary growth curve (stem cell weight) of a heme overproducing strain when grown under aerobic fermentation conditions.

Detailed Description

algae are known to produce a number of compounds that give these aquatic organisms various colors, including but not limited to chlorophyll that turns the algae green, β -carotene that turns the algae yellow or orange, astaxanthin that turns the algae red, or other various pigments such as anthocyanins that turn the algae blue.

Provided herein are strains, methods, and compositions using heme-overproducing algae. In some embodiments, the algal strain is red or a color similar to red when grown. In some embodiments, formulations prepared from algal cultures overproducing heme impart a pink or red color when incorporated into foods and other edible products. In some embodiments, formulations prepared from algal cultures of overproduced heme impart a "meaty" taste, odor, and/or texture when incorporated into food and other edible products.

Without being bound by theory, as shown in fig. 1, the heme pathway is a biochemical pathway that branches from the chlorophyll biochemical pathway. Briefly, this pathway starts with a glutamate tRNA, which is converted to 5-aminolevulinic acid (ALA) by GlutRNA reductase and GSA aminotransferase. Next, ALA is converted to porphobilinogen by ALA dehydratase. Next, porphobilinogen is converted to hydroxymethylcholine by porphobilinogen deaminase. Next, hydroxymethylcholine is converted to uroporphyrinogen III by UPG III synthase. Next, uroporphyrinogen III is converted to coproporphyrinogen (coprophyrinogen) by UPG III decarboxylase. Next, coproporphyrinogen is converted to protoporphyrinogen IX by CPG oxidase. Next, protoporphyrinogen IX is converted to protoporphyrin IX by PPG oxidase. Protoporphyrin IX can be shuttled to the chloral-producing pathway or towards heme B. Finally, protoporphyrin IX is converted to heme B by a ferrochelatase enzyme that attaches iron to protoporphyrin IX.

By reducing metabolic flux towards chlorophyll, metabolic flux towards heme B can be increased. In some embodiments herein, the metabolic flux of algal strains used in the methods and resulting compositions towards chlorophyll is reduced and the metabolic flux towards heme B is increased. In some embodiments, the algal strain is one in which chlorophyll and carotenoid synthesis is reduced and heme synthesis or accumulation is increased. In some embodiments, the algal strain is deficient or reduced in chlorophyll amounts. In some embodiments, the strain of algae is red or a color similar to red.

In some embodiments, the algal strain lacks one or more enzymes of the chlorophyll biosynthetic pathway. These defects include, but are not limited to, deletions, mutations, and other alterations of the gene that result in a lack of expression or functional defect in the enzyme. In some embodiments, the algal strain lacks magnesium chelatase, which is the first step in the conversion of protoporphyrin IX to chlorophyll. In some embodiments, the strain of algae lacks a light-dependent protochlorophyllin, which converts the protochlorophyllin to chlorophyll. In some embodiments, the strain of algae lacks light-independent ortho-chlorophyllin, which converts ortho-chlorophyllin to chlorophyll in the dark. In some embodiments, the algal strain lacks one or more of a ChlB, ChlL, or ChlN gene product. In some embodiments, the algal strain lacks or reduces one or more of a magnesium chelatase, a magnesium protoporphyrinogen IX, a protoporphyrinogen ester, a chlorophyllin ester, and a chlorophyll. In some embodiments, an algal strain lacks one or more of the CHlD1, CHlD2, and CH1H gene products.

In some embodiments, the algal strain overexpresses one or more enzymes such that the balance of the pathway favors heme production. In some embodiments, the algal strain overexpresses one or more of glutamyl-tRNA reductase, glutamyl-1-semialdehyde aminotransferase, ALA dehydrogenase, porphobilinogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelatase. In some embodiments, the ability of an algal strain to produce ALA, a rate-limiting precursor of heme B synthesis, is improved. In some embodiments, the ability of an algal strain to produce a functional ferrochelatase gene responsible for the conversion of protoporphyrin IX to heme B is improved. In some embodiments, the ability of an algal strain to produce UPG III synthase, UPG III decarboxylase, CPG oxidase, or PPG oxidase is improved. In some embodiments, the algal strain has an increased amount of one or more of heme, heme-containing protein, protoporphyrinogen IX, biliverdin IX, phytochrome, and ferrochelatase as compared to a wild-type strain.

exemplary carotenoids include, but are not limited to, gamma-carotene, β cryptoxanthin, zeaxanthin, antherxanthin, lutein, pro-lycopene, and lycopene.

In some embodiments, the strain of algae lacks a carotenoid or a precursor of a carotenoid. Defects in carotenoid biosynthesis may occur due to mutations, such as mutations affecting carotenoid biosynthesis, for example mutations in the lycopene synthase gene.

In some embodiments, algal strains used in the methods herein and for preparing heme-containing compositions are selected or identified based on one or more phenotypes and/or genotypes. In some embodiments, the algal strain used for overproduction of heme can be produced by mating methods. In some embodiments, algal strains used for overproduction of heme can be produced by mutagenesis (such as ultraviolet mutagenesis). In some embodiments, algal strains used for overproduction of heme can be produced by chemical mutagenesis using compounds that cause DNA alteration.

As used herein, the term "genetic modification" is used to refer to any manipulation of genetic material of an organism in a manner that does not occur under natural conditions. Methods for performing such manipulations are known to those of ordinary skill in the art and include, but are not limited to, techniques for transforming cells with a nucleic acid sequence of interest using a vector. Included within the definition are various forms of gene editing, in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases or "molecular scissors". These nucleases generate site-specific Double Strand Breaks (DSBs) at desired locations in the genome. The induced double-strand break is repaired by non-homologous end joining (NHEJ) or Homologous Recombination (HR), resulting in targeted mutation (i.e., editing).

There are several families of engineered nucleases in gene editing, such as, but not limited to, meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas systems, and ARCUS. However, it should be understood that any known gene editing system utilizing engineered nucleases can be used in the methods described herein. Thus, in some embodiments, the heme-overproducing algal strain may be genetically modified by using techniques such as CRISPR-CAS9 or by using zinc finger nucleases.

CRISPR (clustered, regularly interspaced short palindromic repeats) is an acronym for a DNA locus that contains direct repeats of multiple, short base sequences. Prokaryotic CRISPR/Cas systems have been adapted for use as gene editing (silencing, enhancing or altering specific genes) in eukaryotes (see, e.g., Cong, Science,15:339(6121):819-823(2013) and Jinek, et al, Science,337(6096):816-21 (2012)). By transfecting the cell with an element comprising a Cas gene and a specifically designed CRISPR, the nucleic acid sequence can be cleaved and modified at any desired position. Methods of using CRISPR/Cas systems to prepare compositions for genome editing are described in detail in US pub.no.2016/0340661, US pub.no.20160340662, US pub.no.2016/0354487, US pub.no.2016/0355796, US pub.no.20160355797, and WO2014/018423, the entire contents of which are expressly incorporated herein by reference.

Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be designed to target specific desired DNA sequences, which enables zinc finger nucleases to target unique sequences within a complex genome. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genome of higher organisms. The most common cleavage domain is the type IIS enzyme Fok 1. Fok1 catalyzes the double-stranded cleavage of DNA, with a recognition site of 9 nucleotides on one strand and 13 nucleotides on the other. See, e.g., U.S. Pat. nos.5,356, 802; 5,436,150 and 5,487,994; and Li et al Proc., Natl.Acad.Sci.USA 89(1992): 4275-4279; li et al Proc Natl Acad Sci USA 90:2764-2768 (1993); kim et al Proc. Natl. Acad. Sci.USA.91:883-887(1994 a); kim et al.J.biol.chem.269:31,978-31,982(1994b), all of which are incorporated herein by reference. One or more of these enzymes (or enzymatically functional fragments thereof) may be used as a source of cleavage domains.

Methods for selecting algae include, but are not limited to, genetic or phenotypic screens for defects, mutations, and changes in the chlorophyll biosynthesis pathway and/or chlorophyll accumulation, genetic or phenotypic screens for increased expression and/or accumulation of heme, heme biosynthetic intermediates, and heme biosynthetic enzymes. In some embodiments, algal strains used in the methods herein and for making heme-containing compositions are selected or identified based on the spectral distribution of the algal strains and/or the red color or red-like color of the algal strains. In some embodiments, the algae used in the methods herein and for preparing the heme-containing compositions are selected or identified based on the growth rate of the algal strain under dark conditions. In some embodiments, the selection is based on the growth rate under dark conditions and the appearance or enhancement of red or a color similar to red when grown under dark conditions. In some embodiments, strains of algae lacking or having reduced amounts of carotenoids produced or accumulated are selected.

In some embodiments, algal strains are mated to combine or enhance features that contribute to heme production, heme accumulation, chlorophyll reduction, and/or carotenoid reduction. In some embodiments, an algal strain having rapid growth (e.g., faster than a wild-type strain) under dark conditions is mated with an algal strain exhibiting a red color or a color similar to red. In some embodiments, an algal strain lacking carotenoid production or accumulation is mated with an algal strain exhibiting a red color or a color similar to red.

In some embodiments, algal strains are mutagenized, and then new strains are selected or identified that exhibit one or more characteristics of increased heme production, heme accumulation, reduced chlorophyll, and/or reduced carotenoids. In some embodiments, the algal strains are produced by mutagenizing a first starting strain and selecting a second strain that grows faster in the dark than the first starting strain. In some embodiments, the algal strains are produced by mutagenizing a first starting strain and selecting a second strain lacking one or more carotenoids.

Algae for use in compositions and methods

In the compositions and methods provided herein for producing heme and heme-containing compositions, algal strains with heme biosynthetic pathways are used. In some embodiments, the algal line used for overproduction of heme is the phylum Chlorophyta (green alga). In some embodiments, the green algae are selected from the group consisting of: chlamydomonas (Chlamydomonas), Dunaliella (Dunaliella), Haematococcus (Haematococcus), Chlorella (Chlorella), and Scenedesmaceae (Scenedesmaceae). In some embodiments, the Chlamydomonas (Chlamydomonas) is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii). In various embodiments, the green algae can be Chlamydycean, Chlamydomonas (Chlamydomonas), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)137c, or a Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) strain lacking psbA. In some embodiments, the host of choice is Chlamydomonas reinhardtii (Chlamydomonasreinhardtii), such as described in Rasala and Mayfield, Bioeng Bugs, (2011)2(1): 50-4; rasala, et al, Plant Biotechnol j. (2011) May 2, PMID 21535358; coragliotti, et al, MolBiotechnol. (2011)48(1): 60-75; specht, et al, Biotechnol Lett, (2010)32(10): 1373-83; rasala, et al, Plant Biotechnol j. (2010)8(6): 719-33; mulo, et al, BiochimBiophys Acta, (2011) May 2, PMID: 21565160; and Bonente, et al, Photosynth Res. (2011) May 6, PMID: 21547493; US pub.no. 2012/0309939; US pub.no. 2010/0129394; and intl.pub.no.wo2012/170125. All of the foregoing references are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the algal strain used for overproduction of heme is a unicellular alga. Exemplary and additional microalgal species of interest include, but are not limited to: aspergillus orientalis (Achnanthes orientalis), Alternaria gaeuonyssii (Agmenellum), Aphanothece hybrida (Amphiiprora hyaline), Geotrichum coffea (Amphoacoffeiformis), Sphaceloides filiformis (Amphoacoffeiformis Linea), Sphaerotheca coffei var bifidus (Amphoacoffeiformis punctata), Sphaerotheca coffei Tachyta (Amphoacoffeiformis taylori), Sphaerotheca coffei (Amphoa coffeiformis tenuis), Sphaerotheca coffei (Amphoa delatica), Sphaerotheca coffei (Amphoa), Sphaerotheca coffei (Boschoecium), Sphaerotheca (Bochycentrotus), Bochycentrotus heterotheca (Bochycentrotus), Bochycentrotus strain (Bochycentrotus), Bochycentrotus strain Bochycentrotus (Bochycentrotus), Bochycentrotus strain (Bochycentrotus), Bochycentrotus strain Bochycentrotus), Bochycentrotus (Bochycentrotus), Bochytrix gra strain (Bochytrii), Bochytrii (Bochytrii, Bochytrinus), Bochytrix gra strain (Bochytrii, Bochytrix gra strain (Bochytrix (, Chaetoceros sp, Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), Chlorella anomala (Chlorella anatrata), Chlorella Antarctica (Chlorella anattica), Chlorella aureoviridis (Chlorella aureoviridis), Chlorella karya (Chlorella candelilla candel), Chlorella capsulata (Chlorella capsulata), Chlorella dehydrata (Chlorella desacetylate), Chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella pumila (Chlorella emersonii), Chlorella ochroleurochaeta (Chlorella salina), Chlorella fuscogilensis (Chlorella fusca), Chlorella fuscogilytica (Chlorella fuscogilensis) and Chlorella fuscogilytica (Chlorella fusca), Chlorella fuscogilva (Chlorella fuscogilva), Chlorella fusella fuscogilva (Chlorella fusca) and Chlorella fuscogilva (Chlorella fuscogilva), Chlorella fuscogilva (Chlorella fuscogilva) and Chlorella fuscogilva (Chlorella fuscogilva) Chlorella rubra (Chlorella minutissima), Chlorella minutissima (Chlorella minutissima), Chlorella mutant (Chlorella mutanbilis), Chlorella nocturna (Chlorella noctuina), Chlorella bardawil (Chlorella parva), Chlorella photophilus (Chlorella phophila), Chlorella protuberans (Chlorella pringii), Chlorella primula pristina (Chlorella protophyces), Chlorella protothecoides (Chlorella protothecoides), Chlorella regularis (Chlorella regularis) and Chlorella regularis (Chlorella regularis. var. miniata, Chlorella regularis (Chlorella regularis. var. miniata), Chlorella regularis (Chlorella regularis) and Chlorella regularis (Chlorella regularis), Chlorella regularis (Chlorella pyrum, Chlorella regularis (Chlorella regularis), Chlorella regularis (Chlorella regularis, Chlorella regularis (Chlorella), Chlorella regularis (Chlorella pyrenos), Chlorella regularis (Chlorella pyrum), Chlorella regularis (strain (Chlorella regularis), Chlorella regularis (Chlorella regularis, Chlorella regularis (Chlorella), Chlorella regularis (Chlorella pyrenos (Chlorella regularis), Chlorella regularis (Chlorella regularis), Chlorella pyrenos (Chlorella), Chlorella regularis (pyr, Chlorella vulgaris (Chlorella vulgaris), Chlorella vulgaris var vulgaris (Chlorella vulgaris f. tertia), Chlorella vulgaris autotrophic variants (Chlorella vulgaris var. aurothrophylla), Chlorella vulgaris green variants (Chlorella vulgaris var. virilis), Chlorella vulgaris common variants (Chlorella vulgaris var. vulgara), Chlorella vulgaris common variants (Chlorella vulgaris f. tertia), Chlorella vulgaris green variants (Chlorella vulgaris f. virens), Chlorella vulgaris (Chlorella vulgaris f. virens), Chlorella flava (Chlorella vulgaris) yellow (Chlorella xanthella, Chlorella viridans), Chlorella left-handed strain (Chlorella viridis), Chlorella vulgaris (Chlorella viridis), Chlorella viridis, Chlorella vulgaris (Chlorella), Chlorella viridis, Chlorella vulgaris (Chlorella viridis), Chlorella viridis, Chlorella vulgaris, Chlorella viridis, Chlorella vulgaris, Chlorella viridis, Chlorella vulgaris, Chlorella viridis, Chlorella vulgaris, Chlorella vulgaris, Chlorella, Cyclotella cryptica (Cyclotella cryptica), Cyclotella merennii (Cyclotella sp.), Cyclotella sp, Dunaliella bainiensis (Dunaliella bardawil), Dunaliella bifida (Dunaliella bioculata), Dunaliella granular (Dunaliella grandiflora), Dunaliella ocellata (Dunaliella grandiflora) Dunaliella maritima (Dunaliella maritima), Dunaliella ternifera (Dunaliella maritima), Dunaliella bardata (Dunaliella viridis), Dunaliella bardawil (Dunaliella viridis), Dunaliella pellis (Dunaliella viridis), Dunaliella viridis, Dunaliella viridis, Dunaliella, Myxococcus (Gleoocapsas sp.), Rhodococcus (Gloehamomonas sp.), Rhodococcus (Gloeothamnion sp.), Vaniomonas (Hymenomonas sp.), Isochrysis aff galbana), Isochrysis galbana (Isochrysis galbana), Lepocinclis (Lepocinclis), Microastrus (Micrating. sp.), Microastrus (Micrating. LB2614), Micromonospora parvum (Monochrysia minuta), Monochrysis (Monochryshia sp.), Microcystis (Nannochlorus sp.), Microcystis (Nannochloropsis sp.), Microcystis salina (Nannochloropsis), Microcystis microscophyta (Naochlores), Microcystis (Naochlorus), Microcystis (Naochloa), Novophora (Naringoniella), Novophora (Naochloa), Novophora (Naringoniensis), Novophora), Novophyceae (Naringoniella), Novophyceae (Naringi), Novophyceae (Naringi), Novophyceae, Naringoniella (Naringi), Naringoniella (Naringi), Naringi (Naringi), Naringoniella phyceae), Naringi (Naringoniella (Naringi), Naringi, Naringoniella naphalophyceae, Nitzschia disipata (Nitzschia disipata), Nitzschia fruticosa (Nitzschia fruticosa), Nitzschia hantzeri (Nitzschia hantzschia), Hippocampus (Nitzschia incosporicularia), Nitzschia intermedia (Nitzschia intermedia), Nitzschia microcystis (Nitzschia microcephala), Nitzschia microcephala (Nitzschia pusilla), Nitzschia ellipsoidea (Nitzschia pusilla), Nitzschia macroalgae (Nitzschia quadrata), Nitzschia sp, Physiomonas sp, Phormidium chrysosporium (Ochromonas sp), Physiomonas sp), Physiocladia sp, Phormidis (Ochromonas sp), Physiomonas sp, Physiocladia sp, Physiomonas sp, Physiosphaeria monocytoglosa, Physiosphaera (Oscilaria sp), Physiosphaera sp), Physiosphaeria sp, Physiosphaeria monocytoglosa sp, Physiosphaeria sp, Physiomonas sp, Physiosphaeria sp, Physio, Coccolithospermum dentatum (Pleurochrysis dentata), Coccolithospermum (Pleurochrysis sp.), Prototheca welsonii (Protothecaherchamii), Prototheca calophylla (Prototheca stagnora), Prototheca nodosa (Prototheca portoriensis), Prototheca sanguinea (Prototheca mori), Prototheca fuliginosum (Prototheca magna), Pyrococcus sp, Morus (Pyrobotrys), Chrysophyces capsulatus (Sarcodysophystis), Scenedesmus (Scenedesmus armatus), Schizochytum (Schizochytrium), Spirogyra, Spirulina platensis (Spirotensis), Schizochyta (Chlorococcus), Tetrasella sp (Tetrasella sp), Tetrasella sp). In some embodiments, the algae are of the species Chlamydomonas (Chlamydomonas). In some embodiments, the algae is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii). In some embodiments, the algae are derivatives of a strain of Chlamydomonas viridis (Chlamydomonas) prepared by mutagenesis or by mating with another strain of algae.

Method for culturing heme overproducing strains

Methods for culturing algae in liquid culture media include a variety of options including ponds, ditches, small scale laboratory systems, and closed and partially closed bioreactor systems. Algae can also be grown directly in water, for example, in oceans, seas, lakes, rivers, reservoirs, and the like.

In some embodiments, heme overproduced algae useful in the methods and compositions provided herein are grown in controlled culture systems (such as small scale laboratory systems, large scale systems, and/or closed and partially closed bioreactor systems). Small scale laboratory systems refer to cultures that are less than about 6 liters in volume and can range from about 1 milliliter or less to about 6 liters. Large scale culture refers to a culture having a volume greater than about 6 liters, and can range from about 6 liters to about 200 liters, and even larger scale systems having a footprint of 5 to 2500 square meters or more. The large scale culture system may comprise a liquid culture system of about 10000 to about 20000 liters and up to about 1000000 liters.

Culture systems for use with methods for producing the compositions herein include closed structures (such as bioreactors) in which the environment is more tightly controlled than open or semi-closed systems. Photobioreactors are bioreactors that incorporate some type of light source to provide a photonic energy input to the reactor. The term bioreactor may refer to a system that is closed from the environment and does not exchange gases and/or contaminants directly with the environment. The bioreactor can be described as a closed, culture vessel designed to control biomass production of liquid cell suspension cultures in the case of photobioreactor illumination.

In some embodiments, the algae used in the methods and compositions provided herein are grown in a fermentation vessel. In some embodiments, the vessel is a stainless steel fermentation vessel. In some embodiments, the algae are grown under heterotrophic conditions, thereby providing one or more carbon sources to the culture. In some embodiments, the algae are grown under aerobic and heterotrophic conditions. In some embodiments, the algae is grown to a density of greater than or about 10g/L, about 20g/L, about 30g/L, about 40g/L, about 50g/L, about 75g/L, about 100g/L, about 125g/L, or about 150 g/L.

In some embodiments, the algae is inoculated from the seed tank to an initial density of greater than about 0.1g/L, about 1.0g/L, about 5.0g/L, about 10.0g/L, about 20.0g/L, about 50g/L, about 80g/L, or about 100 g/L. Once inoculated, the algae grow heterotrophically using aerobic fermentation methods. In this method, algae is fed nutrients to maintain their growth. In some embodiments, these nutrients include a source of reducing carbon. Exemplary sources of reducing carbon include, but are not limited to, acetate, glucose, sucrose, fructose, glycerol, and other sugars. In some embodiments, the algal culture is supplemented with iron.

In some embodiments, the algae are cultured under dark conditions. In some embodiments, the algae cultured under dark conditions lack or reduce chlorophyll production. In some embodiments, the algae grown under dark conditions are supplemented with one or more nutrients. In some embodiments, algae grown under dark conditions are grown in the presence of a reducing carbon source (e.g., acetate, glucose, sucrose, fructose, glycerol, and other sugars). In some embodiments, algae grown under dark conditions are grown or otherwise supplemented with iron in the presence of iron.

Heme-containing preparations and products

Algal strains and heme-overproducing cultures such as those described herein can be used in various forms and formulations. In some embodiments, the heme-containing composition is prepared from an algal culture that overproduces heme, wherein the composition is red or a color similar to red.

In some embodiments, the heme-containing composition is prepared from biomass isolated from cultured algae. In some embodiments, the biomass is further stratified to remove one or more constituents. In some embodiments, the biomass is layered to remove starch. In some embodiments, the biomass is stratified to remove proteins. In some embodiments, the biomass is stratified or otherwise treated to remove carotenoids. In some embodiments, the biomass is layered or otherwise treated to enrich certain components. In some embodiments, the layered or treated biomass is heme-rich. In some embodiments, the layered or treated biomass is enriched in proteins or in proteins and heme. In some embodiments, layering or treating enhances the red color or a color similar to red of the formulation. The layered or treated biomass may be enriched in protein content such that the composition is about 10% protein, greater than about 10% protein, or greater than about 20%, about 30%, about 40%, or about 50% protein.

In some embodiments, the heme-containing composition is a heme-containing liquid prepared from a culture medium of a cultured algae. In some embodiments, the heme-containing composition is prepared from heme found extracellularly in an algal culture. In some embodiments, the algae culture is lysed or otherwise treated to release heme from the cells. In some embodiments, the heme-containing liquid is further layered to remove one or more components. In some embodiments, the heme-containing liquid is layered to remove starch. In some embodiments, the heme-containing liquid is layered to remove proteins. In some embodiments, the heme-containing liquid is layered or otherwise treated to remove carotenoids. In some embodiments, the heme-containing liquid is stratified or otherwise treated to enrich certain components. In some embodiments, the layered or treated heme-containing liquid is heme-rich. In some embodiments, layering or treating enhances the red color or a color similar to red of the formulation.

Heme-containing compositions, including biomass, liquids, and layered preparations, may be further processed. Such processing may include concentration, drying, lyophilization and freezing. In various embodiments, the heme-containing composition can be combined with additional ingredients and ingredients. In some embodiments, the heme-containing composition is combined with additional ingredients to produce an edible product. In some embodiments, the heme-containing composition imparts a red color or a color similar to a red color to the edible product. In some embodiments, the heme-containing composition imparts a meat-like characteristic (such as a meat-like taste, aroma, and/or texture) to the edible product.

In some embodiments, the heme-containing composition is combined with additional ingredients to produce a meat-like product. Such meat-like products may include clean meat or cultured meat (made from animal cells grown in the laboratory or otherwise outside the animal), vegetable-based meat, and non-animal-based meat (made from vegetable ingredients and/or ingredients of non-animal origin). In some embodiments, the heme-containing composition made from the overproduced algae is combined with additional ingredients to produce a meat-like product, whereby the addition of the heme-containing composition imparts a red or red-like color, a meat-like aroma, a meat-like taste, and/or a meat-like texture to the meat-like product. In some embodiments, the meat-like characteristics imparted by the heme-containing composition are imparted to a raw or uncooked product. In some embodiments, the meat-like characteristics imparted by the heme-containing composition are imparted to the cooked product.

Examples

Example 1: identification of algae overproducing heme

Strains of algae (Chlamydomonas reinhardtii) that overexpress heme were identified by their inability to produce chlorophyll. In addition, these strains exhibit red, brown, orange or some variants of the listed colors. The identified strain exhibits photosensitivity, andcannot be more than 10 mu E m^-2s^-1The growth is carried out for a long time under direct light.

One of the identified strains was grown under aerobic fermentation conditions in fed-batch culture, in which acetate was used as a reducing carbon source for the culture nutrients. Strains were grown in fermenters where minimal light could reach the culture. The lines were grown to a density of greater than 50g/L and harvested by centrifugation. The harvested lines are red and can be added to the composition (approved stock) to impart red, orange or brown color. FIG. 3 is a graph showing the cell weight of a strain of heme overproducers grown under aerobic fermentation conditions.

Example 2: layering

Cells from a heme-overproducing strain of Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) are harvested from the fermentation culture. The harvested cells were disrupted by sonication, and then the sample was separated by centrifugation at 10000 XG. This separates the sample into carotenoid, starch and protein/heme biomass components. The protein/heme biomass was then resuspended in phosphate buffered saline at ph 7.4. Demixing after centrifugation (left) and resuspension with heme-containing components (right) are shown in fig. 2.

Example 3: characterization of heme production

Many heme assays can be used to determine the concentration of heme. In one embodiment, the amount of heme can be quantitatively determined by: the algal biomass is mixed into an aqueous alkaline solution, thereby converting heme to a uniform color. Color intensity can be measured by absorbance at 400nm, which is proportional to the concentration of hemoglobin in the sample. These measurements can then be compared to standards produced from known concentrations of hemoglobin to determine the amount of hemoglobin in the algae sample.

Example 4: preparation of heme-rich edible products

The heme-rich sample can be used to prepare compositions of meat-like products produced from plant-based materials and heme-rich algae. In this example, 20 grams of coconut oil, 20 grams of texturized wheat protein, 53 grams of water, 5 grams of algal biomass rich in heme, 1 teaspoon of konjac gum, 1 teaspoon of xanthan gum, and 1 gram of yeast extract were mixed and formed into a discoid, algal plant based hamburger. After cooking, algae-based hamburgers can bleed like meat-based hamburgers.

Claims (70)

1. A composition suitable as a food ingredient, the composition comprising a preparation from an algal strain that overexpresses heme.

2. The composition of claim 1, wherein the formulation is biomass from an algal strain.

3. The composition of claim 2, wherein the formulation is a layered biomass from an algal strain.

4. The composition of claim 3, wherein the layered biomass comprises a heme-rich component.

5. The composition of claim 4, wherein the heme-rich component further comprises a protein-rich component.

6. The composition of claim 1, wherein the agent is an extracellular component of an algal culture.

7. The composition of any one of claims 1-6, wherein the preparation is purified from a protein.

8. The composition of any one of claims 1-7, wherein the formulation imparts a red color or a color similar to red to the composition.

9. The composition of any one of claims 1-8, wherein the formulation imparts a meat or meat-like taste to the composition.

10. The composition of any one of claims 1-9, wherein the formulation imparts a meat or meat-like texture to the composition.

11. The composition of any one of claims 1-10, wherein the algae is Chlamydomonas (Chlamydomonasp.).

12. The composition of claim 11, wherein the Chlamydomonas is Chlamydomonas reinhardtii (Chlamydomonasreinhardtii).

13. A food product comprising a heme-containing formulation according to any one of claims 1 to 12.

14. The food product of claim 13, wherein the food product comprises clean meat, cultured meat, synthetic meat, plant-based meat, or non-animal cell-based meat.

15. A method of producing a heme composition, the method comprising: growing a culture of algae, wherein the algae are heme overproducers; and isolating the heme composition from the culture.

16. The method of claim 15, wherein the algae are grown by aerobic fermentation.

17. The method of claim 15 or 16, wherein said algae comprise chloroplasts.

18. The method of any one of claims 15-17, wherein heme biosynthesis occurs in a chloroplast.

19. The method of any one of claims 15-18, wherein the algae lacks the ability to produce chlorophyll.

20. The method of any one of claims 15-19, wherein said algae lacks the ability to produce functional Mg-chelatase.

21. The method of any one of claims 15-20, wherein said algae lacks a functional ChlD1, ChlD2, or ChlDH gene product.

22. The method of any one of claims 15-21, wherein said algae lacks functional light-dependent protochlorophyllin.

23. The method of any one of claims 15-22, wherein the algae lacks functional light-independent ortho-chlorophyllin.

24. The method of any one of claims 15-23, wherein said algae lacks a functional ChlB, chl, or ChlN gene product.

25. The method of any one of claims 15-24, wherein algae overexpresses a gene selected from the group consisting of: glutamyl-tRNA reductase, glutamyl-1-semialdehyde transaminase, ALA dehydrogenase, porphobilinogen deaminase, UPGIII synthase, UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelatase.

26. The method of any one of claims 15-25, wherein the algae are produced by mating, and wherein the resulting strain is red or a color similar to red.

27. The method of any one of claims 15-26, wherein the algae is produced by mutagenesis.

28. The method of any one of claims 15-27, wherein the algae is red or a color similar to red.

29. A method of any one of claims 15-28, wherein the isolated heme composition is algal biomass.

30. The method of claim 29, wherein the isolated heme composition is a stratified algal biomass.

31. The method of claim 30, wherein the isolated heme composition is a protein-rich stratified algal biomass.

32. The method of any one of claims 15-28, wherein the isolated heme composition is isolated from the extracellular medium of the algal culture.

33. A method of any of claims 15-32, wherein the isolated heme composition is isolated from an algal protein.

34. The method of any one of claims 15-33, wherein the algae is deficient in carotenoids.

35. The method of any one of claims 15-34, wherein the algae is Chlamydomonas.

36. The method of any one of claims 15-35, wherein the algae is chlamydomonas reinhardtii.

37. A method as claimed in any one of claims 15 to 36, wherein the heme-overexpressed algal strain is produced by mating a strain that grows faster in the dark than wild-type algae under the same conditions with a strain that already exhibits a red or red-like color.

38. A method according to any one of claims 15-36, wherein the heme overproducer is a strain produced by mating a carotenoid-deficient strain with a strain exhibiting a red color or a color similar to red.

39. A method of any one of claims 15-36, wherein the heme overproducer is produced by mutagenesis of a first starting line and selection of a second line that grows faster in the dark than the first starting line.

40. A method according to any one of claims 15-36, wherein said overproducer of heme is produced by mutagenesis of a first starting strain and selection of a second strain lacking one or more carotenoids.

41. A product comprising a heme composition produced by the method of any of claims 15-40.

42. The product of claim 41, wherein the heme composition imparts a red color or a red-like color to the product or portion thereof.

43. A product as claimed in claim 41 or 42, wherein the heme composition imparts a meat or meat-like flavour to the product or part thereof.

44. A product according to any of claims 41-43, wherein the heme composition imparts a meat or meat-like texture to the product or a portion thereof.

45. Heme purified from an algal strain produced by the method of any one of claims 15-40.

46. The method of any one of claims 15-40, wherein said algae is deficient in carotenoids.

47. A method of producing a heme-containing composition, the method comprising:

(a) culturing an algal strain under dark conditions, wherein the strain produces no chlorophyll or reduced chlorophyll production; and

(b) collecting a portion of the algal culture.

48. The method of claim 47, wherein the algae is Chlamydomonas.

49. The method of claim 48, wherein said algae is Chlamydomonas reinhardtii.

50. The method of any one of claims 47-49, wherein said algae has increased protoporphyrinogen IX.

51. The method of any one of claims 47-50, wherein said algae exhibits a red color or a color similar to red when grown under said dark conditions.

52. The method of any one of claims 47-51, wherein the collected portion of the algal culture exhibits a red color or a color similar to a red color.

53. The method of any one of claims 47-52, wherein the collected fraction is extracellular medium from the algal culture.

54. The method of any one of claims 47-53, wherein the collected fraction is biomass from the algal culture or stratified biomass.

55. The method of any one of claims 47-54, wherein said algal strain lacks one or more functional gene products selected from the group consisting of: ChlD, ChlD2, CHlH, light-activated protochlorophyllide oxidoreductase and light-independent protochlorophyllide gene products.

56. The method of any one of claims 47-55, wherein the algae are grown by aerobic fermentation.

57. The method of any one of claims 47-56, wherein the algae is grown to a density of greater than about 10g/L, about 20g/L, about 30g/L, about 40g/L, about 50g/L, about 75g/L, about 100g/L, about 125g/L, or about 150 g/L.

58. The method of any one of claims 47-57, wherein the algae are grown using acetate as a reducing carbon source.

59. The method of any one of claims 47-58, wherein the algae are grown using sugar as a reducing carbon source.

60. The method of any one of claims 47-59, wherein the algal culture is supplemented with iron during the culturing step.

61. The method of any one of claims 47-60, wherein the algal culture is inoculated at a density of greater than about 0.1g/L, about 1.0g/L, about 5.0g/L, about 10g/L, about 20g/L, about 50g/L, about 80g/L, or about 100 g/L.

62. The method of any one of claims 47-61, further comprising stratifying the collected fraction, wherein said stratifying removes substantially all or most carotenoids from the collected fraction.

63. The method of any one of claims 47-62, further comprising layering the collected portion, wherein the layering removes substantially all or most of the starch from the collected portion.

64. The method of any one of claims 47-63, further comprising stratifying the collected fraction, wherein the stratifying produces a protein-rich fraction.

65. The method of any one of claims 47-64, wherein the algal strain has increased ferrochelatase expression as compared to a wild type algal strain.

66. The method of any one of claims 47-65, wherein the algal strain has an increased amount of heme, heme-containing protein, protoporphyrinogen IX, biliverdin IX, plant pigment, ferrochelatase, or any combination thereof.

67. The method of any one of claims 47-66, wherein the algal strain lacks one or more of a magnesium chelatase, magnesium protoporphyrinogen IX, a chlorophyllin ester, and chlorophyll.

68. The cleaned meat product produced by the method of any one of claims 47-67, wherein the method further comprises mixing the collected portion with a composition made from cleaned meat, wherein the collected portion provides a red color or a red-like color to the cleaned meat product.

69. The cleaned meat of claim 68, wherein the collected portion is a heme-rich component or a purified heme.

70. The method of any one of claims 47-67, wherein the algal strain is not a transgenic strain.

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