BIO-based production of n-acetylcysteine

Abstract

Disclosed herein are methods for the fermentative production of N-acetylcysteine (NAC) or NAC-containing products from unicellular organisms. Also disclosed herein are genetic modifications of the NAC and / or substrate biosynthetic pathways in unicellular organisms that include bacteria, algae, microalgae, diatoms, yeast, or fungi. Further disclosed herein are fermentation and processing methods for the production of various NAC-containing products. The present disclosure further relates to use of the cells, fermentation broth or extracts that contain NAC to produce products for use in food, feed, beverages, dietary and health supplements, cosmetics, personal care, pharmaceuticals, or agricultural production.

Description

[0001] BIO-BASED PRODUCTION OF N-ACETYLCYSTEINE CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 688,626 filed on August 29, 2024, the entirety of which is hereby incorporated by reference for all purposes. SEQUENCE SUBMISSION

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled Bio-Based Production of N-Acetylcysteine.xml, created on June 25, 2025 and is 19,499 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in their entirety. FIELD OF THE INVENTION

[0003] The present invention is in the field of production of N-acetylcysteine (NAC) by unicellular organisms. BACKGROUND

[0004] N-acetylcysteine

[0005] N-acetyl-cysteine (NAC) is naturally occurring in living organisms. NAC is a powerful antioxidant that effectively scavenges reactive oxygen species (ROS). As a precursor to glutathione, the body’s most vital antioxidant, NAC is crucial for neutralizing free radicals and protecting cells from oxidative damage. The level of NAC in some plant species is reported to be high, specifically Allium.[1] NAC is reported to be synthesized from glutathione but a glutathione pathway has not been explicitly identified. There are reports of NAC produced as an intermediate step as part of a S-(2-succino) cysteine (2SC) detoxification system that is present in some bacteria. [2-4]

[0006] NAC is currently manufactured using petroleum feedstocks in a multi-step chemical synthesis process. The key step in manufacturing NAC involves the acetylation of L-cysteine, which is done by reacting L-cysteine with acetic anhydride (ethanoic anhydride) or acetyl chloride. The acetylating agents, acetic anhydride and acetyl chloride, are hazardous and toxic substances. A real need exists for a biologically synthesized, safe and sustainable source of NAC that can be economically produced on a commercial scale.

[0007] The present invention provides methods for a cost-effective fermentative production of NAC by unicellular organisms. Methods are presented for the optimization of NAC production through genetic improvements of unicellular organisms, growth and fermentation conditions, cost- effective nutrient media and downstream processing for NAC purification.

[0008] S-(2-succino) cysteine catabolic pathway

[0009] In the cell, elevated levels of the tricarboxylic acid (TCA) cycle intermediate, fumarate, can spontaneously react with cysteine to form S-(2-succino) cysteine (2SC). Several groups have shown that 2SC can be broken down into cysteine and acetate through a three-step catabolic pathway by components in the yxe operon (Figure 1). [2-4] The first gene in the operon, yxeL, encodes for an enzyme that controls the N-acetylation of 2SC to form N-acetyl- 2SC (2SNAC). The second gene in the operon, yxeK, encodes for an FAD-dependent monooxygenase, yxe, that coverts 2SNAC into oxaloacetate and NAC. The last gene in the operon, N-acetylcysteine deacetylase, yxeP, converts NAC into cysteine and acetate.

[0010] Cysteine Cysteine production requires carbon, nitrogen and sulfur. Methods to increase cysteine have been developed and demonstrated in microbes. Carbon and nitrogen are supplied from components of the serine-based pathway. Sulfur (sulfate and thiosulfate) is supplied to the cell through a series of reactions that involve uptake, reduction and assimilation. Carbon from glucose enters the serine biosynthetic pathway by conversion of glycerate1,3-bisphosphate into glycerate 3- phosphate by the pgk gene product phosphoglycerate kinase. Glycerate 3-phosphate is converted into 3-phosphohydroxypyruvate by the product of serA, 3-phosphoglycerate dehydrogenase. The serA gene product is sensitive to feedback inhibition by serine, however, the inhibition can be removed by the deletion of the last 197 amino acids (serA∆197) of the serA gene product from Corynebacterium glutamicum or by the 3 amino acid changes in the serA gene product from Escherichia coli (serAfbr). 3-phosphohydroxypyruvate is converted into O-phospho-serine by the product of serC, phosphoserine aminotransferase, and O-phospho-serine is converted into serine by the product of serB, phosphoserine phosphatase. Serine and acetyl-CoA are converted into O- acetyl-serine by the product of cysE, serine acetyltransferase. The cysE gene product is sensitive to feedback inhibition by cysteine, however, a mutated cysEM201Ris insensitive to cysteine inhibition. O-acetyl-serine is converted into cysteine by the product of cysK, cysteine synthase. Cysteine can be degraded by the product of tna. Other serine-based precursors are derived from the above-named compounds. The precursor, 2-aminoacrylate, is produced from serine by threonine dehydratase, a product of ilvA or serine dehydratase. The ilvA gene product is sensitive to feedback inhibition by isoleucine, however, a mutated ilvAL447F is insensitive to isoleucine inhibition. 2-aminoacrylate is converted into 2-ketobutyate by the products of RidA or tdcF, 2- iminobutanoate / 2-iminopropanoate deaminases or the product of rutC, aminoacrylate peracid reductase. Decreased demand for serine and increased availability of serine precursors can be accomplished by diverting serine from other pathways through the decreased activities of the gene products for glyA, ilvA , sdaA, sdaB, tdcG, and entC.

[0011] Sulfur Metabolism

[0012] Sulfur-based precursors for cysteine and NAC biosynthesis come from the sulfur uptake and reduction pathways. Methods to increase cysteine have been developed and demonstrated in microbes. [5, 6] The sulfate-thiosulfate uptake pathway is controlled by the products of sbp, cysP, cysU, cysW, and cysA. Sulfate and thiosulfate are bound by the products of sbp and cysP, respectively, and transported into the cell by the products of cysU, cysW, and cysA.[7] Sulfate is converted into 3’- phosphoadenosine‐5′‐phosphosulfate (PAPS) by the products of cysDNC, ATP sulfurylase and APS kinase. PAPS is converted into adenosine‐3′,5′‐diphosphate (PAP) and sulfite by the product of cysH, PAPS reductase. The product of cysQ, PAP nucleotidase, is involved in PAPS regeneration. Sulfite is converted into sulfide by the products of cysIJ. O- acetyl-L-serine and sulfide are converted into cysteine by CysK and CysM. CysM also synthesizes S-sulfocysteine from O-acetyl-L-serine and thiosulfate. The S-sulfocysteine is converted into cysteine by glutaredoxin (NrdH) or Grx.

[0013] Fumarate

[0014] Several methods to increase fumarate have been developed and demonstrated in microbes. [8, 9] One approach to increase fumarate levels is to increase carbon flow into the TCA cycle by increasing the expression of key enzymes in the pathway such as pyruvate carboxylase (pyc), citrate synthase (cs); aconitase (acn) isocitrate lyase (aceA) and succinate dehydrogenase (sdh).

[0015] Another method to increase fumarate is to minimize the formation of by-products: such as lactate, by knocking out lactate dehydrogenase (ldhA); acetate, by knocking out pyruvate oxidase (poxB), phosphotransacetylase (pta), or acetate kinase A (ackA); formate, by knocking out pyruvate formate lyase (pflB); malate, by knocking out fumarase (fumABC); and succinate, by knocking out fumarate reductase (frdBC).

[0016] Others approaches to increase fumarate include the use of a noncyclic glyoxylate cycle

[0010] and altering parts of the urea cycle and the purine nucleotide cycle.

[0011]

[0017] Fermentation Conditions and Nutrient Media

[0018] In the described invention NAC is produced by fermentation. Methods to produce chemical compounds by batch fermentation, fed-batch fermentation, continuous fermentation or in tanks or ponds are well known to one with ordinary skill in the art.[12-22]

[0019] The culture medium to be used in the present invention is dependent upon the requirements of the microorganism used in production. Descriptions of defined media for various microorganisms are found in the literature.[23-25] Carbon sources can be used individually or combined and can include sugar and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, fatty acids, alcohols, and organic acids and glycerol. Nitrogen sources can be used individually or as a mixture and can include organic nitrogen-containing compounds such as peptones, tryptone, casein amino acids, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Potassium and phosphate sources can include potassium chloride, monopotassium phosphate, dipotassium phosphate, monosodium phosphate, and disodium phosphate. Magnesium sulfate or iron sulfate, micronutrients, amino acids and vitamins are also necessary for growth.

[0020] To control the pH of the culture, compounds such as sodium hydroxide, potassium hydroxide, ammonia, ammonium hydroxide or acids such as phosphoric acid or sulfuric acid are used. To control the foaming, anti-foaming agents are used. Aerobic conditions are maintained by mixing or introducing air or oxygen into the culture. The dissolved oxygen is 15% to 40%, depending on the growth phase and microorganism. The temperature of the culture is 25°C to 40° C, preferably at 30°C to 37°C, depending on the microorganism. Growth of the cell culture is maintained until maximum NAC production is reached, typically within 10 hours to 100 hours, preferably 15 hours to 30 hours.

[0021] In the described invention, the fermentation broth contains NAC, the cell mass of the microorganism, organic by-products of the fermentative process, and any remaining components of the medium.

[0022] The concentration of NAC synthesized can be determined at various times throughout fermentation using high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrospray ionization mass spectrometry (ESI-MS), and liquid chromatography tandem mass spectrometry (LC–MS / MS).

[0023] Downstream Processing: Separation and Purification

[0024] In the described invention NAC is processed or purified to make a product. The specific downstream processing to be used is dependent upon several factors including whether NAC exists in the cells (or biomass) or in the liquid, the form of the desired final NAC product such as liquid or powder, and the desired purity and / or moisture level. In some product applications, the processing may include drying the cells and media to the appropriate concentration and dryness. In some product applications, the processing may include purifying or partially purifying the NAC. To decrease cost and increase efficiency, the volume can be decreased at various times throughout downstream processing by concentrating or removing water by evaporation, using e.g. a falling film evaporator, reverse osmosis or nanofiltration.

[0025] If the NAC is in the liquid of the fermentation broth, the liquid can be separated from the biomass by centrifugation, filtration, decantation or a combination thereof. Additional processing of the NAC-containing liquid may include concentration or drying or a purification step for the manufacturing of a NAC product according to the invention. The purification step may be selected from the group consisting of chromatographic techniques or membrane-based processes including ion exchange chromatography, ultra-filtration, precipitation, pH adjustment and nanofiltration, treatment with activated carbon or crystallization. The purification step or any combination thereof may be repeated until the NAC is purified to the desired specification such as for purity and moisture.

[0026] If the NAC is in the cells of the fermentation broth, the cells can be separated from the liquid by centrifugation, filtration, decantation or a combination thereof. The NAC-containing cells can be concentrated and used as a product or the cells can be disrupted by chemical agents, pressure, mechanical force, or ultrasonification to release their contents. The disrupted cells with their contents can be concentrated or dried and used as a product or the contents can be further processed to produce single cell proteins that can be concentrated or dried for use as a product. Alternatively, NAC in the disrupted cells can be separated from the cellular debris by centrifugation, filtration or decantation or a combination thereof, followed by further purification as described above.

[0027] If the NAC is in both the liquid and the cells in the fermentation broth, the liquid and cells can be separated, and treated separately, as described above or concentrated together. The NAC- containing concentrate can be used for the manufacturing of a product according to the invention or further processed by purification as described above.

[0028] The NAC-containing product can be in different forms such as liquid, powder, paste, capsule or tablet. SUMMARY

[0029] The invention provides methods for the fermentative production of NAC-containing products in unicellular organisms. More particularly, the invention encompasses the use of polynucleotides for NAC biosynthetic enzymes in combination with polynucleotides to increase cysteine and fumarate as precursors for NAC production in the cell or for the secretion of NAC out of the cell into the media.

[0030] The invention also relates to fermentation and processing methods for the production of various products produced from the cells, fermentation broth or extracts that contain NAC.

[0031] For purposes of promoting an understanding of the principles of the invention, reference will now be made to particular embodiments of the invention and specific language will be used to describe the same. The materials, methods and examples are illustrative only and not limiting.

[0032] In some embodiments, the unicellular organisms contain one or more exogenous polynucleotides that is operably linked to a promoter. In other embodiments, the expression of the endogenous polynucleotides of the unicellular organisms is modified with an exogenous promoter.

[0033] In one embodiment, the invention consists of unicellular organisms that have an yxe operon that expresses the endogenous polynucleotides, yxeL and yxeK, and contains a knoocked out yxeP gene.

[0034] In another embodiment, the invention consists of unicellular organisms that have a NAC biosynthetic pathway containing the exogenous polynucleotides, yxeL and yxeK, and a modified cysteine pathway to have increased expression of pgk, serA∆197(or serAfbr), serC, serB, cysE, and cysK, and a modified sulfur-based pathway to have increased expression of cysPUWA, cysDNC, cysQ, cysH and cysIJ.

[0035] In another embodiment, the invention consists of unicellular organisms that have a NAC biosynthetic pathway containing the exogenous polynucleotide, yxeL and yxeK, and a modified cysteine pathway to have increased expression of pgk, serA∆197(or serAfbr), serC, and serB, and a modified sulfur-based pathway to have increased expression of cysDNC and cysQ, combined with a modified fumarate pathway with increased expression of one or more of the following genes: pyruvate carboxylase (pyc), citrate synthase (cs); aconitase (acn) isocitrate lyase (aceA) and succinate dehydrogenase (sdh); or knockouts for one or more of the following genes: ldhA, poxB, pta, ackA, pflB, fumABC, or frdBC.

[0036] In another embodiment, the invention consists of unicellular organisms that have a NAC biosynthetic pathway with one or more genes silenced or down regulated to decrease the diversion of serine into other biosynthetic pathways. These may include one or more of the following: glyA, sdaA, sdaB, ilvA, tdcG , tdcG and entC.

[0037] The invention includes modified or mutant unicellular organisms including bacteria, yeast, fungi, or unicellular algae that produce NAC for use in food, feed, beverages, dietary and health supplements, cosmetics, personal care, pharmaceuticals, or agricultural production.

[0038] The invention also describes methods to grow the cells by fermentation and describes media formulations in which to grow the cells for the production of NAC or a NAC-containing product that may be a liquid, powder, paste, capsule or tablet.

[0039] The invention also describes methods to process the cells or the media in which the cells were grown to make a range of products that include pure NAC or a NAC-containing product. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 exemplifies the pathway for NAC production in a unicellular organism. Elevated levels of fumarate react with the thiol group of cysteine to produce S-(2-succino)-L-cysteine (2SC) through a spontaneous Michael addition reaction. The yxeL gene product acetylates 2SC to produce N-acetyl-S-(2-succino)-L-cysteine (NAC2SC). The yxeK gene product converts NAC2SC into oxaloacetate and N-acetyl-L-cysteine (NAC). Genes are designated in bold italicized text, and molecules are in normal text. DETAILED DESCRIPTION

[0041] The present invention provides methods for the production of NAC in unicellular organisms. In preferred embodiments, the invention provides methods for the genetic modification of unicellular organisms using genes that encode proteins in the NAC biosynthetic pathway, the cysteine biosynthetic pathway, and increase fumarate. The invention also provides methods of using unicellular organisms including bacteria, microalgae, fungi, yeast, and algae with increased levels of endogenous NAC for use in food, feed, beverages, dietary and health supplements, cosmetics, personal care, pharmaceuticals, or agricultural production.

[0042] This invention presents methods for the modification of unicellular organisms that contain an yxe operon that overexpress the endogenous polynucleotides, yxeL and yxeK and the corresponding functional peptides combined with an yxeP knockout.

[0043] This invention presents methods for the modification of unicellular organisms that increase the expression of exogenous polynucleotides for yxeL and yxeK and the corresponding functional peptides.

[0044] This invention presents methods for the modification of unicellular organisms that increase the expression of one or more polynucleotides for peptides in cysteine pathways comprising of: pgk, serA∆197(or serAfbr), serC, serB, cysEM201R, cysK, cysM, nrdH, sbp, cysUWA, cysPUWA, cysDNC, cysQ, cysH, and cysIJ.

[0045] This invention presents methods for the modification of unicellular organisms with increased levels of fumarate by increased expression of one or more of the following genes: pyruvate carboxylase (pyc), citrate synthase (cs); aconitase (acn) isocitrate lyase (aceA) and succinate dehydrogenase (sdh).

[0046] This invention presents methods for the modification of unicellular organisms with increased levels of fumarate by methods of silencing, mutating or knocking out one or more of the following genes: ldhA, poxB, pta, ackA, pflB, fumABC, or frdBC.

[0047] Below is a non-limiting list of example polynucleotides that are suitable for of the invention. Other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of polynucleotides by selective hybridize to the polynucleotides to the named polypeptide by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Still other suitable polynucleotides for use in accordance with the invention may be obtained by the identification of similar polynucleotides that have substantial identity of the nucleic acid of or encode polypeptides that have substantial identity to amino acid sequence of when it used as a reference for sequence comparison.

[0048] Suitable polynucleotides for yxeL are provided in SEQ ID NO:1; SEQ ID NO:3 and encode the peptides with amino acid sequences of SEQ ID NO:2; SEQ ID NO:4, respectively.

[0049] Suitable polynucleotides for yxeK are provided in SEQ ID NO:5; SEQ ID NO:7 and encode the peptides with amino acid sequences of SEQ ID NO:6; SEQ ID NO:8, respectively.

[0050] Suitable polynucleotides for yxeK are provided in SEQ ID NO:9; SEQ ID NO:11 and encode the peptides with amino acid sequences of SEQ ID NO:10; SEQ ID NO:12, respectively.

[0051] The invention is not limited to the use of these amino acid sequences. Amino acid sequences comprising of the variation of the enzymes and transcription factors listed are included within the scope of the present invention and are considered substantially or sufficiently similar to a reference amino acid sequence. Although it is not intended that the present invention be limited by any theory by which it achieves its advantageous result, it is believed that the identity between amino acid sequences that is necessary to maintain proper functionality is related to maintenance of the tertiary structure of the polypeptide such that specific interactive sequences will be properly located and will have the desired activity, and it is contemplated that a polypeptide including these interactive sequences in proper spatial context will have activity.

[0052] One of ordinary skill in the art will recognize that changes in the amino acid sequences, such as individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is “sufficiently similar” when the alteration results in the substitution of an amino acid with a chemically similar amino acid.

[0053] It is therefore understood that the invention encompasses more than the specific polynucleotides encoding the proteins described herein. For example, modifications to a sequence, such as deletions, insertions, or substitutions in the sequence, which produce “silent” changes that do not substantially affect the functional properties of the resulting polypeptide are expressly contemplated by the present invention.

[0054] It is understood that alterations in a nucleotide sequence, which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.

[0055] When the nucleic acid is prepared or altered synthetically, one of ordinary skill in the art can take into account the known codon preferences for the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in different species, sequences can be modified to account for the specific codon preferences and GC-content preferences of the organism, as these preferences have been shown to differ.

[0026]

[0056] Cloning Techniques

[0057] Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. Specific terms, while employed below and defined at the end of this section, are used in a descriptive sense only and not for purposes of limitation. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, mycology, phycology, tissue culture, molecular biology, chemistry, biochemistry, biotechnology, and recombinant DNA technology, which are within the skill of the art.

[0027]

[0058] A suitable polynucleotide for use in accordance with the invention may be obtained by cloning techniques using cDNA or genomic libraries, DNA, or cDNA from bacteria, algae, microalgae, diatoms, yeast or fungi. Furthermore, nucleic acid sequences may be constructed or amplified using chemical synthesis. The product of amplification is termed an amplicon. Moreover, if the particular nucleic acid sequence is of a length that makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments that may be synthesized and ligated together to form the entire desired sequence by methods known in the art. Alternatively, individual components or DNA fragments may be amplified by PCR and adjacent fragments can be amplified together using fusion-PCR, overlap-PCR or chemical (de novo) synthesis using a vendor (e.g. DNA2.0, GE life technologies, GENEART, Gen9, GenScript) by methods known in the art.

[0059] The recombinant expression cassette or DNA construct includes a promoter that directs transcription in a unicellular organism, operably linked to the polynucleotide of the invention described herein. A variety of different types of promoters are described and used. As used herein, a polynucleotide is "operably linked" to a promoter or other nucleotide sequence when it is placed into a functional relationship with the promoter or other nucleotide sequence. The functional relationship between a promoter and a desired polynucleotide insert typically involves the polynucleotide and the promoter sequences being contiguous such that transcription of the polynucleotide sequence will be facilitated. Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1) result in the introduction of a frame-shift mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region. Typically, the promoter element is generally upstream (i.e., at the 5′ end) of the nucleic acid insert coding sequence.

[0060] While a promoter sequence can be ligated to a coding sequence prior to insertion into a vector, in other embodiments, a vector is selected that includes a promoter operable in the host cell into which the vector is to be inserted. In addition, certain preferred vectors have a region that codes a ribosome binding site positioned between the promoter and the site at which the DNA sequence is inserted so as to be operatively associated with the DNA sequence of the invention to produce the desired polypeptide, i.e., the DNA sequence of the invention in-frame.

[0061] Gene expression cassettes may contain one or more polynucleotides (genes), each operably linked with a promoter and terminator to form a series of monocistronic mRNAs or the genes can be arranged with one promoter and terminator to form a single polycistronic mRNA. A wide variety of operable cassettes are known to those of ordinary skill in the art.

[0062] Suitable Promoters

[0063] A wide variety of promoters are known to those of ordinary skill in the art, as are other regulatory elements that can be used alone or in combination with promoters. A wide variety of promoters that direct transcription in unicellular organisms can be used in connection with the present invention.

[0028] The features (binding sites and regulatory elements) necessary for the identification and use of functional bacterial promoters are known to those of ordinary skill in the art

[0029] For purposes of describing the present invention, promoters are divided into two types, namely, constitutive promoters and non-constitutive promoters. Constitutive promoters are classified as providing for a range of constitutive expression. Some are weak constitutive promoters, and others are strong constitutive promoters. Other promoters are considered non- constitutive promoters.[30-32]

[0064] Terminators

[0065] In addition to the selection of a suitable promoter, the DNA constructs require an appropriate transcriptional terminator to be attached downstream (3’), after the stop codon (TGA, TAG or TAA) of the desired gene of the invention for proper expression in unicellular organisms. Several such terminators are available and known to persons of ordinary skill in the art. Terminators play an important role in the processing and stability of RNA as well as in translation and may also control gene expression.[33, 34] The identification and use of terminators that are required to express genes in unicellular organisms are known to those of ordinary skill in the art.

[0066] Suitable Vectors

[0067] A wide variety of vectors may be employed to transform a unicellular organism with a construct made or selected in accordance with the invention, including high- or low-copy number plasmids, phage vectors and cosmids. Vector systems, expression cassettes, culture methods, and transformation methods are known by those of ordinary skill in the art. The vectors can be chosen such that operably linked promoter and polynucleotides that encode the desired polypeptide of the invention are incorporated into the genome of the unicellular organism. Other vectors that can operably link promoter and polynucleotides that encode the polypeptide of the invention are incorporated are not incorporated into the host genome but the vector DNA with the clone polynucleotides are autonomously or semi autonomously replicated in the cell. Although the preferred embodiment of the invention is expressed in unicellular organisms, other embodiments may include expression in prokaryotic or unicellular eukaryotic organisms including, but not limited to, yeast, fungi, algae, microalgae, or microbes.

[0068] It is known by those of ordinary skill in the art that there exist numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. There are many commercially available recombinant vectors to transform a unicellular organism. Standard molecular and cloning techniques[27, 35] are available to make a recombinant expression cassette that expresses the polynucleotide that encodes the desired polypeptide of the invention. No attempt will be made to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes. In brief, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter, followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high-level expression of a cloned gene, it is desirable to construct expression vectors that contain, at the minimum, a strong promoter, to direct transcription, a ribosome-binding site for translational initiation, and a transcription / translation terminator.

[0069] Expression in Prokaryotes

[0070] Protocols for transformation as well as commonly used vectors with control sequences including promoters for transcription initiation (some with an operator), together with ribosome binding site sequences for use in prokaryotes are known to those of ordinary skill in the art. Those of ordinary skill in the art know the molecular techniques and DNA vectors that are used in bacterial systems.[36, 37] In bacteria one messenger RNA can encode for one peptide (referred to as monocistronic) or several independent peptides (referred to as polycistronic). It is known to those of ordinary skill in the art that a portion of a polycistronic messenger RNA can be knocked-out

[0038] or that heterologous or exogenous genes can be expressed on a monocistronic or polycistronic messenger RNA.

[0037] Genes can be expressed by modification of bacterial DNA (genomic) through the use of knock-in, gene insertion, or by allelic exchange.[39, 40] Specific gene targeting has been used in bacteria using PCR-based methods,

[0041] and CRISPR / Cas.

[0042]

[0071] Transformation of Host Cells

[0072] Transformation of an unicellular organism can be accomplished in a wide variety of ways within the scope of a person of ordinary skill in the art.[28, 43-45] Those of ordinary skill in the art can use different algal, diatom, fungal, yeast and bacteria gene transfer techniques [46, 47] Other methods to edit, incorporate or move genes into bacteria, fungal algal genomes include, but are not limited to, Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats / Cas (CRISPR / Cas).

[0073] Gene Silencing by Mutagenesis or Recombinant Technologies

[0074] Genetic modification to silence or inactivate genes or their corresponding gene products of unicellular organisms can be conducted by radiation-, chemical- or UV-based mutagenesis followed by specific screening for biochemical traits or pathways.

[0048] Radiation-based mutations can silence or inactive a gene or the corresponding gene product by DNA breakage and repair. Chemical- or UV-based mutations usually result in single DNA basepair changes. Mutations can silence or inactive a gene or the corresponding gene product by one of the following: (1) introduction of a frame-shift mutation; (2) introduction of premature stop codon; (3) interference with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, (4) interference with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region or (5) introduction of an amino acid substitution in the gene product to reduce or inhibit activity (enzymatic activity or binding) or interfere with the function of the gene product.

[0075] Methods for targeted gene silencing or knockouts can be made in unicellular organisms using known by those of ordinary skill in the art,

[0049]

[0050] ,

[0051]

[0039]

[0052]

[0053]

[0054] including use of CRISPER-Cas9 or CRISPi

[0055] In addition, RNA-mediated methods,

[0056] or regulatory RNAs

[0057] can be used to silence or suppress gene expression in unicellular organisms and these techniques and protocols are well known to one with ordinary skill in the art.

[0076] Suitable Unicellular Organisms

[0077] A wide variety of unicellular host cells may be used in the invention, including prokaryotic and unicellular eukaryotic host cells. These cells or organisms may include yeast, fungi, algae, microalgae, microbes, or unicellular photosynthetic organisms. Preferred host cells for this invention are bacteria including, archaebacteria and eubacteria. Proteobacteria such as members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria can host the invention. Other bacteria including Methanotrophs and Methylobacterium can be used with the invention. Other bacterial genera that can host the invention include, but are not limited to Escherichia, Bacillus, Salmonella, Lactococcus, Lactobacillus, Streptococcus, Brevibacterium and Coryneform bacteria. Some specific bacterial species that can be used for the invention include, but are not limited to, Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterium pekinese, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum, Gluconobacter oxydans, Propionibacterium freudenreicheii, Propionibacterium denitrificans, and Saccharomyces cerevisiae.

[0078] Pharmaceutical Compositions

[0079] Pharmaceutically acceptable vehicles of NAC are tablets, capsules, gel, ointment, film, patch, powder or dissolved in liquid form.

[0080] Nutritional Supplements and Feeds

[0081] Transgenic cells containing NAC may be consumed or used to make extracts for nutritional supplements. Transgenic cells that contain NAC may be used for human consumption. Extracts from transgenic cells containing NAC may be used as nutritional supplements, as an antioxidant or to improve physical or mental performance. The extracts may be used in the form of a liquid, powder, capsule or tablet.

[0082] Enhancer of Plant Growth or Yield

[0083] Transgenic cells that contain NAC may be used as an enhancer for seed germination, plant growth or yield. Extracts from transgenic cells containing NAC may be used as plant enhancers in the form of a liquid, powder, capsule or tablet. DEFINITIONS

[0084] The term “microbe” refers to any microorganism (including both eukaryotic and prokaryotic microorganisms), such as bacteria, fungi, yeast, bacteria, algae and protozoa, as well as other unicellular organisms.

[0085] The terms “polypeptide,” “peptide,” “protein” and “gene product” are used interchangeably herein to refer to a polymer of amino acid residues. Amino acids may be referred to by their commonly known three-letter or one-letter symbols. Amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0086] The terms “residue,” “amino acid residue,” and “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide. The amino acid may be a naturally occurring amino acid and may encompass known analogs of natural amino acids that can function in a similar manner as the naturally occurring amino acids.

[0087] The term “yxeL gene product” refers to S-(2-succino)cysteine N-acetyltransferase, a protein that catalyzes the following reaction: acetyl-CoA + S-(2-succino)-L-cysteine = CoA + H++ N-acetyl-S-(2-succino)-L-cysteine NOTE: yxeL is also referred to as scmL.

[0088] The term “yxeK gene product” refers to N-acetyl-S-(2-succino)cysteine monooxygenase, a protein that catalyzes the following reaction: H++ N-acetyl-S-(2-succino)-L-cysteine + NADH + O2 = H2O + N-acetyl-L-cysteine + NAD++ oxaloacetate NOTE: yxeK is also referred to as scmK. NOTE: N-acetyl-S-(2-succino)cysteine monooxygenase is also referred to as FAD-dependent monooxygenase

[0089] The term “yxeP gene product” refers to N-acetylcysteine deacetylase, a protein that catalyzes the following reaction: H2O + N-acetyl-L-cysteine = acetate + L-cysteine NOTE: yxeP is also referred to as scmPL.

[0090] The term “vector” includes reference to a nucleic acid used in transfection or transformation of a host cell and into which can be inserted a polynucleotide.

[0091] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.”

[0092] Methods of alignment of nucleotide and amino acid sequences for comparison are well known to those of ordinary skill in the art. The local homology algorithm, BESTFIT,

[0058] can perform an optimal alignment of sequences for comparison using a homology alignment algorithm called GAP,

[0059] search for similarity using Tfasta and Fasta,

[0060] by computerized implementations of these algorithms widely available on-line or from various vendors (Intelligenetics, Genetics Computer Group). CLUSTAL and other software allows for the alignment of multiple sequences

[0061]

[0062] The BLAST family of programs can be used for nucleotide or protein database similarity searches.

[0093] GAP

[0059] maximizes the number of matches and minimizes the number of gaps in an alignment of two complete sequences. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.

[0063]

[0094] Unless otherwise stated, sequence identity or similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters.

[0064] As those of ordinary skill in the art understand that BLAST searches assume that proteins can be modeled as random sequences and that proteins comprise regions of nonrandom sequences, short repeats, or enriched for one or more amino acid residues, called low-complexity regions. These low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. Those of ordinary skill in the art can use low-complexity filter programs to reduce number of low-complexity regions that are aligned in a search.[65, 66]

[0095] The terms “sequence identity” and “identity” are used in the context of two nucleic acid or polypeptide sequences and include reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conserved substitutions, the percent sequence identity may be adjusted upwards to correct for the conserved nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have “sequence similarity” or “similarity.” Scoring for a conservative substitution allows for a partial rather than a full mismatch,

[0067] thereby increasing the percentage sequence similarity.

[0096] The term “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise gaps (additions or deletions) when compared to the reference sequence for optimal alignment. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0097] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 50-100%.

[0098] The terms “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm

[0059] . Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conserved substitution. Another indication that amino acid sequences are substantially identical is if two polypeptides immunologically cross-react with the same antibody in a western blot, immunoblot or ELISA assay. In addition, a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical.

[0099] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention known by those of ordinary skill in the art.

[0100] The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the References.

[0101] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

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Claims

CLAIMS 1. A method for NAC production by a unicellular organism wherein the organism produces at least 0.1 g / liter of NAC when grown in a shaker flask or 1 g / liter of NAC when grown in a fermentor or bioreactor.

2. The method of claim 1 wherein the unicellular organism expresses one or more exogenous NAC biosynthetic pathways and contains one or more of the following: a. increased expression of a gene in the serine biosynthetic pathway; b. increased expression of a gene in the cysteine biosynthetic pathway: c. increased expression of a gene in the TCA cycle: d. increased expression of genes in sulfate or thiosulfate transport, or sulfur reduction or sulfur assimilation; e. deletion of a gene involved in lactate, acetate, formate, malate or succinate biosynthesis; or f. deletion of a gene involved in NAC deacetylation.

3. The method of claim 1, wherein the unicellular organism is selected from the group consisting of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Methanotrophs, Methylobacterium, Escherichia, Bacillus, Salmonella, Lactococcus, Lactobacillus Streptococcus, Brevibacterium, coryneform bacteria, Bacillus subtilis, Brevibacterium ammoniagene, Corynebacterium crenatum, Corynebacterim pekinese, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Escherichia coli, Fusarium venenatum,Gluconobacter oxydans, Propionibacterium freudenreicheii, Propionibacterium denitrificans and Saccharomyces cerevisiae.

4. The method for isolating NAC by the method of claim 1 wherein NAC has a purity level of the following: a. >10% pure; b. >25% pure; c. >50% pure; d. >75% pure; or e. >98% pure.

5. The method of claim 1, wherein the unicellular organism is E. coli, grown in a media that contains at least 5 g / L ammonium sulfate, 6 g / L dibasic potassium phosphate, 3 g / L monobasic sodium phosphate, 0.5 g / L magnesium sulfate, 6 g / L glucose, 0.1g / L typtone and 0.05g / L yeast extract.

6. The method of claim 1, wherein the cells of the unicellular organism are chemically, physically or mechanically disrupted, dried and used in food, feed, beverages, dietary and health supplements, cosmetics, personal care, pharmaceuticals, or agricultural production.

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