Method for producing carotenoids from acid whey

Abstract

Disclosed herein are metabolically engineered cells capable of producing carotenoids from acid whey.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 304,412, filed January 28, 2022, entitled "METHOD OF PRODUCING CAROTENOIDS FROM ACID WHEY," the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (M065670522WO00-SEQ-KVC.xml; size: 83,140 bytes; and creation date: January 19, 2023) are incorporated herein by reference in their entirety.

[0003] Field Provided herein are methods and compositions relating to the production of carotenoids from acid whey. [Background technology]

[0004] background Acid whey (AW) is a liquid waste by-product that, if disposed of untreated, poses serious environmental problems due to its high organic matter content. It is primarily produced in the production of Greek yogurt, which is becoming increasingly popular in the United States, but there are other large sources worldwide, such as paneer production. It is estimated that over 3 million tonnes of AW are produced annually in the United States. This represents an economic burden to the dairy industry, but at the same time offers a valuable opportunity that could be used to produce valuable products. Summary of the Invention

[0005] overview The present disclosure relates, at least in part, to methods and compositions for producing carotenoids from acid whey. Aspects of the present disclosure relate to modified yeast cells capable of producing carotenoids from acid whey. In some embodiments, the present disclosure relates to modified yeast cells capable of converting acid whey to pyruvate via either lactate metabolism, glucose metabolism, or galactose metabolism via the Leloir pathway. In some embodiments, the present disclosure relates to modified yeast cells capable of converting pyruvate to lycopene via the mevalonate pathway. In some embodiments, the present disclosure relates to modified yeast cells capable of converting lycopene to astaxanthin and / or lutein. In some embodiments, the modified yeast cells described herein are modified to overcome substrate inhibition. Aspects of the present disclosure relate to genetically engineered yeast cells (modified cells) that include: a heterologous gene, where the heterologous gene encodes an enzyme having β-galactosidase (LacA) activity; one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate; one or more heterologous genes encoding one or more enzymes of the Leloir pathway; and one or more heterologous genes encoding one or more enzymes of the mevalonate pathway. In some embodiments, the modified cell is an oleaginous yeast cell. In some embodiments, the oleaginous cell is a Yarrowia lipolytica cell.

[0006] In some embodiments, the one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate are selected from the group consisting of lactate transporter (JEN1) and lactate dehydrogenase (LDH). In some embodiments, the one or more heterologous genes encoding one or more enzymes of the Leloir pathway are selected from the group consisting of GAL10M, GAL1, GAL7, and GAL10E. In some embodiments, the one or more heterologous genes encoding one or more enzymes of the mevalonate pathway are selected from the group consisting of GGPPS, CarRP, and CarB. In some embodiments, the GGPPS is GGPPSxd from Xanthophyllomyces dendrorhous, GGPPSsa from Sulfolobus acidocaldarius, GGPPStc from Taxus canadensis, GGPPSpa from Pantoea agglomerans, and GGPPSyl from Yarrowia lipolytica.

[0007] In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having lycopene β-cyclase activity. In some embodiments, the enzyme having lycopene β-cyclase activity comprises an amino acid sequence at least 90% identical to the amino acid sequence represented by SEQ ID NO:1. In some embodiments, the enzyme having lycopene β-cyclase activity comprises an amino acid sequence represented by any one of SEQ ID NOs:2-4. In some embodiments, the modified cell further comprises a heterologous gene encoding tHMGR, ERG12, IDI, and ERG20 of the mevalonate (MVA) pathway, and / or choline kinase (CK) and isopentenyl phosphate kinase (IPK). In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having β-carotene ketolase (CrtW) activity and a heterologous gene encoding an enzyme having β-carotene hydroxylase (CrtZ) activity. In some embodiments, the enzyme having CrtW activity is fused to an enzyme having CrtZ activity. In some embodiments, the CrtW / CrtZ fusion enzyme comprises a localization signal. In some embodiments, the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisomes, and / or fat bodies.

[0008] In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having lycopene β-cyclase activity and / or a heterologous gene encoding an enzyme having lycopene ε-cyclase activity. In some embodiments, the enzyme having lycopene β-cyclase activity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having carotenoid hydroxylase 1 (LUT1) activity and / or a heterologous gene encoding an enzyme having carotenoid hydroxylase 5 (LUT5) activity.

[0009] In some embodiments, the modified yeast cells described herein can overcome substrate inhibition. As used herein, the term "substrate inhibition" refers to the most common deviation from Michaelis-Menten kinetics, which occurs in about 25% of known enzymes. Substrate inhibition occurs when the concentration of the enzyme substrate exceeds the optimal parameters and reduces the growth rate of the cell.

[0010] Another aspect of the present disclosure relates to a genetically engineered yeast cell (modified cell) comprising: a first heterologous gene, where the first heterologous gene encodes an enzyme having β-carotene ketolase (CrtW) activity; and a second heterologous gene, where the second heterologous gene encodes an enzyme having β-carotene hydroxylase (CrtZ) activity; where the modified cell produces β-carotene. In some embodiments, the modified cell is an oleaginous yeast cell. In some embodiments, the oleaginous cell is a Yarrowia lipolytica cell. In some embodiments, the enzyme having CrtW activity is fused to an enzyme having CrtZ activity. In some embodiments, the CrtW / CrtZ fusion enzyme comprises a localization signal. In some embodiments, the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisome, and / or fat body.

[0011] Another aspect of the present disclosure relates to a method of converting a carbon source to lycopene and / or β-carotene, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient time to convert the carbon source to lycopene and / or β-carotene. In some embodiments, the carbon source is acid whey. In some embodiments, the carbon source is converted to lycopene. In some embodiments, the carbon source is converted to β-carotene.

[0012] Another aspect of the present disclosure relates to a method of converting a carbon source to astaxanthin, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient time to convert the carbon source to astaxanthin. In some embodiments, the carbon source is acid whey.

[0013] Another aspect of the present disclosure relates to a method of converting a carbon source to alpha-carotene, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient time to convert the carbon source to alpha-carotene. In some embodiments, the carbon source is acid whey.

[0014] Another aspect of the present disclosure relates to a method of converting a carbon source to lutein, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient period of time to convert the carbon source to lutein. In some embodiments, the carbon source is acid whey.

[0015] Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 2. Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 3. Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 4.

[0016] Each of the limitations of the present invention may encompass various aspects of the present invention. Thus, it is anticipated that each of the limitations of the present invention, including any one element or combination of elements, may be included in each aspect of the present invention. The present invention is not limited in its application to the details of the configuration and arrangement of components set forth in the following description or illustrated in the drawings. The present invention can be practiced or carried out in other embodiments and in various ways. Additionally, the phraseology and terminology used in this disclosure are for purposes of explanation and should not be considered limiting. The use of "including," "including," or "having," "containing," "involving," and variations thereof in this disclosure means the inclusion of the items listed thereafter and their equivalents, as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0017] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims. [Brief description of the drawings]

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale. The drawings are merely illustrative and are not required to enable the present disclosure. For clarity, not every component has been labeled in every drawing. In the drawings:

[0019] [Figure 1A-1C]Figure 1A, Consumption of sugars and organic acids present in AW by the engineered Y. lipolytica strain (Lac, lactose; Glu, glucose; Gal, galactose). Figure 1B, Production of cell biomass and lipids during fermentation. Figure 1C, Fatty acid composition of yeast cells. [Diagram 2] Metabolic pathway for carotenoid synthesis from lycopene.

[0020] [Diagram 3] Overview of metabolic engineering efforts required for the biosynthesis of lycopene from AW using Y. lipolytica. Lactose hydrolysis involved the introduction of β-galactosidase (LacA), enhanced lactate to pyruvate conversion involved overexpression of lactate transporter (JEN1) and dehydrogenase (LDH), enhanced native galactose metabolism was achieved by overexpression of Leloir pathway genes, and lycopene biosynthesis involved the introduction of three heterologous genes: geranylgeranyl diphosphate synthase (GGPPxd), phytoene synthase (CarRP), and phytoene desaturase (CarB).

[0021] [Figure 4]Engineered intracellular astaxanthin biosynthetic pathway in Yarrowia lipolytica. Cytosolic acetyl-CoA was the common precursor for lipid formation and astaxanthin synthesis. β-carotene synthesized in the endoplasmic reticulum (ER) was sequestered into lipid bodies (LBs) aggregated from triacylglycerides (TAGs). The stored TAGs were hydrolyzed to free fatty acids (FFAs), which were transported to peroxisomes with β-carotene translocation and converted to acetyl-CoA by β-oxidation. The genes involved in the β-carotene biosynthetic pathway previously engineered in Y. lipolytica are GGPPsa, CarRP, and CarB. CrtW / Z (CrtW and CrtZ) are the heterologous intracellular engineered enzymes related to astaxanthin biosynthesis in this study. GGPPsa, geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius; CarRP, bifunctional phytoene synthase / lycopene β-cyclase from Mucor circinelloides; CarB, phytoene dehydrogenase from M. circinelloides; CrtW, β-carotene ketolase; CrtZ, β-carotene hydroxylase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; FBP, fructose 1,6-bisphosphatase; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; PA, phosphatidic acid; DAG, diacylglycerol. Dotted arrows indicate multiple catalytic steps.

[0022] [Figure 5A-5B]Optimization of astaxanthin production. Figure 5A, an astaxanthin biosynthetic pathway starting from β-carotene was constructed by introducing β-carotene ketolase (CrtW) and hydroxylase (CrtZ). Expression of the pathway was accompanied by a change in colony color from yellow to red. Figure 5B, Maximization of astaxanthin production by testing CrtW and CrtZ from diverse organisms. With the optimal combination of PsCrtW from Paracoccus sp. and HpCrtZ from Haematococcus pluvialis, maximum astaxanthin production was obtained after 72 h of shake flask cultivation. Below each bar, various construct combinations are shown (filled circles are included, blank columns are not). The mean and standard deviation (sd) of three independent experiments are shown.

[0023] [Figure 6A-6B] Biosynthesis of astaxanthin by various fusion constructs in Y. lipolytica. Figure 6A, Astaxanthin biosynthesis pathway. Depending on the order of ketonization and hydroxylation effected by CrtW and CrtZ, respectively, multiple routes to astaxanthin from β-carotene are possible, resulting in the formation of different intermediates (indicated by numbers). CrtW is β-carotene ketolase; CrtZ is β-carotene hydroxylase. Figure 6B, HPLC traces of carotenoids from flask cultures of astaxanthin-producing strains expressing fusion CrtW-Z or individual enzymes CrtW+Z. Compared to the YL02 strain harboring individual enzymes CrtW+Z, the accumulation of canthaxanthin ((4)) and zeaxanthin ((6)) in the fusion CrtW-Z strain was significantly reduced, whereas the intermediate 3'-hydroxyechinenone ((5)) was accumulated at a higher level. [Figure 6C]Figure 6C, Accumulation of astaxanthin in strains harboring functional fusion enzymes. Compared to the control strain expressing the individual enzymes CrtW+Z, the astaxanthin levels of the strains expressing the fusion enzymes were significantly increased. The CrtW-Z fusion strain showed even more enhanced performance than the CrtZ-W fusion. Linker sequence, GGGGSGGPGS (SEQ ID NO: 5). The average and deviation of three independent experiments are shown.

[0024] [Figure 7A] Subcellular organelle engineering further promoted astaxanthin biosynthesis. Figure 7A, Schematic diagram of astaxanthin biosynthesis when the fusion enzyme CrtW-Z-dependent pathway was targeted to the endoplasmic reticulum (ER), fat body (LB), and peroxisome by fusion with KDEL, oleosin, and SKL sequences, respectively. [Figure 7B-7C] Figure 7B, chromatographic carotenoid profile of organelle-targeted strains. Compared to strains with cytosolic expression, accumulation of intermediates, especially 3'-hydroxyechinonone ((5)), echinonone ((2)) and β-carotene ((1)), was significantly reduced in strains with two (ER and LB) or three (ER, LB and peroxisome) organelle manipulations. Figure 7C-7D, assembly of the astaxanthin pathway into intracellular organelles further accelerated the conversion of β-carotene to astaxanthin. [Figure 7D] Figure 7C-7D, assembling the astaxanthin pathway into intracellular organelles further accelerated the conversion of β-carotene to astaxanthin. Furthermore, simultaneously targeting the fusion enzyme CrtW-Z to all three organelles, i.e., LB, ER, and peroxisomes, resulted in the highest astaxanthin titers after 72 h of shake flask culture in YPD medium. The different construct combinations were shown below each bar (+, with organelle targeting; -, without organelle targeting). The mean and standard deviation of three independent experiments were shown. Statistical differences were analyzed using Student's t-test, and P<0.05 was considered statistically significant. ***P<0.001; **P<0.01.

[0025] [Figure 8A] The strain engineered for astaxanthin production achieved high titers in fed-batch cultures. Figure 8A, Astaxanthin production by strain YL17 cultured in YPD medium containing different initial glucose concentrations (20, 30, 40, and 50 g / L). [Fig. 8B-8C] 8B-8C, Fed-batch fermentation profiles of astaxanthin-producing strain YL17 in conical flasks (FIG. 8B) and 3-L bioreactors (FIG. 8C). The average and standard deviation of three independent experiments are shown.

[0026] [Figure 9] HPLC analysis of carotenoids in fusion strains harboring CrtZ-W or CrtW-Z: (1), β-carotene; (2), echinenone; (4), canthaxanthin; (5), 3'-hydroxyechinenone; (6), zeaxanthin; (9), astaxanthin. [Figure 10A] Figure 10A, Effect of additional copies of the fusion enzyme CrtW-Z on astaxanthin production in engineered strains. Each plus (+) sign indicates one copy of the gene integrated into the genome. The average and standard deviation of three independent experiments are shown. [Figure 10B] FIG. 10B. HPLC analysis of carotenoids in engineered strain YL12 harboring an additional copy of the fusion enzyme CrtW-Z: (1), β-carotene; (2), echinenone; (4), canthaxanthin; (5), 3′-hydroxyechinenone; (6), zeaxanthin; and (9), astaxanthin.

[0027] [Figure 11] Microscopic image of β-carotene-producing cells. Large amounts of β-carotene were clearly stored in the fat bodies of the cells. [Figure 12A-12B] Prediction of the transmembrane helices of the β-carotene biosynthetic enzymes GGPPsa (Figure 12A), CarRP (Figure 12B), and CarB (Figure 12C) using the TMHMM server v. 2.0. [Figure 12C] Prediction of the transmembrane helices of the β-carotene biosynthetic enzymes GGPPsa (Figure 12A), CarRP (Figure 12B), and CarB (Figure 12C) using the TMHMM server v. 2.0. [Figure 13] Subcellular localization of the β-carotene biosynthetic enzymes GGPPsa, CarRP, and CarB by fusion with GFP protein.

[0028] [Figure 14] Chromatographic carotenoid profiles of engineered YL17 strains grown in various media. The unique difference between the media was the initial concentration of glucose: YPD20, 20 g / L glucose; YPD30, 30 g / L glucose; YPD40, 40 g / L glucose; YPD50, 50 g / L glucose. (1), β-carotene; (2), echinenone; (4), canthaxanthin; (5), 3'-hydroxyechinenone; (6), zeaxanthin; (9), astaxanthin. [Figure 15] Correlation between dry cell weight (DCW) and OD600 in astaxanthin-producing cells. DCW was calculated based on the measured OD600 and by applying a conversion factor.

[0029] [Figure 16A] Lycopene cyclase showed a substrate inhibition effect. Figure 16A, Lycopene inhibited the downstream enzyme, lycopene cyclase, by substrate inhibition. As a result, if the rate of lycopene formation is higher than the rate of its subsequent conversion to β-carotene, the imbalance may be aggravated, leading to the accumulation of lycopene. [Fig. 16B-16C] (B) After fermentation in YPD medium for 3 days, β-carotene levels in strains expressing related biosynthetic genes from different sources showed that the CarB / CarRP pair reached a higher level of performance. (C) Heterologous overexpression of GGPP synthase (GGPPxd) from X. dendrorhous increased β-carotene production but also led to the massive accumulation of its biosynthetic precursor, lycopene. (ND) Not detected. [Figure 16D] Figure 16D, Relative lycopene cyclase catalytic activity measurements showed that the activity of wild-type CarRP (or CarR) was biphasic with respect to lycopene concentration, indicating substrate inhibition. In contrast, CarRP (or CarR) variant Y27R had no substrate inhibition at all. CarR, truncated CarRP without the P domain. In Figures 16B-16D, the average and standard deviation (sd) of three independent experiments were shown.

[0030] [Figure 17A] Ablation of substrate inhibition by protein engineering. Figure 17A. Using predictive protein models, several positions within the R domain of CarRP (lycopene cyclase) were identified as suitable sites for mutation to reduce substrate inhibition. Single substitutions are indicated by light spheres and double substitutions by dark spheres. [Fig. 17B-17C] In Figure 17B, β-carotene selectivity was tested for a total of 50 variants generated. Compared to the wild type (WT), Y27R, V175W, and T31R-F92W (boxed) showed a significant increase in β-carotene selectivity, suggesting reduced substrate inhibition. Data represent the average of two independent experiments. In Figure 17C-17D, compared to the control strain YLMA03 harboring wild-type CarRP, the variants showed a significant increase in β-carotene production, along with a decrease in lycopene accumulation (Figure 17C). [Fig. 17D-17E] In particular, the YLMA11 strain expressing CarRP (Y27R) achieved a titer of 2.38 g / L (Figure 17C) with a high selectivity of 98% (Figure 17D). Figure 17E, loss of substrate inhibition allowed a higher flux through the carotenoid synthesis pathway (via overexpression of MVA and IUP), improving β-carotene titer while maintaining high selectivity. Finally, 4.22 g / L of β-carotene was produced in YLMA15 with approximately 98% selectivity. For cultures with strains containing IUP, 30 mM isoprenol (Figure 31) was added to the medium after glucose depletion. For Figures 17C and 17E, the average and standard deviation of three independent experiments are shown.

[0031] [Figure 18A-18B] GGPPS-mediated metabolic flow restrictor effectively relieved substrate inhibition. Figure 18A, GGPPS-mediated metabolic flow restrictor altered the amount of flux through the carotenoid synthesis pathway and regulated the rate of lycopene formation. Figure 18B, Changes in GGPPS activity can be achieved by expressing enzymes from different organisms in Y. lipolytica, as shown by changes in the rate of GGPP synthesis in vivo. These experiments used the po1f background strain with no modifications other than GGPPS expression. [Fig. 18C-18D] FIG. 18C, Compared with the strain expressing GGPPxd, other strains carrying low activity GGPPS alleviated the substrate inhibition effect of lycopene cyclase. The slow formation rate of lycopene prevented lycopene accumulation, and almost all lycopene was converted to β-carotene. ND, not detected. FIG. 18D, The fermentation time course showed that the balanced pathway with attenuated GGPPsa (YLMA25) minimized lycopene accumulation throughout the experiment, which was consistent with YLMA11 containing the Y27R variant of CarRP. On the contrary, rapid lycopene accumulation was observed in the strain with highly efficient GGPPxd (YLMA03). [Fig. 18E-18G] Figure 18E, gene expression cassettes containing balanced pathways (GGPPsa, CarB, and CarRP) were sequentially introduced into the po1f-T strain for β-carotene production. With higher copy numbers, the β-carotene titer increased to 2.13 g / L, maximizing the selectivity for β-carotene. Figure 18F, overexpression of MVA and IUP further improved β-carotene synthesis while maintaining its high selectivity. Figure 18G, substrate inhibition was deliberately induced using the highly efficient GGPPxd, together with the mutated CarRP(E78K), to construct a lycopene-producing strain that reached a titer of 2.62 g / L. ND, not detected. For Figures 18B-18G, the average and standard deviation of three independent experiments are shown.

[0032] [Figure 19A]Balancing acetyl-CoA distribution between lipid and isoprenoid synthesis benefited carotenoid accumulation. Figure 19A, cytosolic acetyl-CoA was shared between the two competing pathways, i.e., de novo lipid biosynthesis and the MVA pathway. However, intracellular lipid bodies formed hydrophobic regions where carotenoids could be sequestered, facilitating their accumulation. As a result, both pathways were required, and optimal partitioning of flux was essential to achieve high carotenoid titers and contents per cell. Moreover, intracellular TAG could be used as a carbon source for acetyl-CoA generation, which in turn provides the building blocks for carotenoids. [Fig. 19B-19D] Figure 19B, lipid content was dependent on the C / N ratio of the medium, with higher C / N ratios promoting lipid production. Figures 19C-19D, β-carotene titer (Figure 19C) and content (Figure 19D) were also functions of the C / N ratio of the medium. However, unlike lipid content, which increased monotonically with C / N ratio, there was an optimum for β-carotene production. The highest β-carotene titer and content per cell were obtained at a C / N ratio of 9:1 in Y10P10D50 medium. For Figures 19B-19D, the average and standard deviation of three independent experiments are shown.

[0033] [Figure 20A-20B] Cellular lipids promoted carotenoid biosynthesis via β-oxidation during the stationary phase after glucose depletion. In Figure 20A, monitoring the glucose concentration, lipid content, and β-carotene content during fermentation revealed that β-carotene continued to increase even after glucose was exhausted from the medium. At the same time, intracellular lipids rapidly decreased after glucose depletion, suggesting that cells mobilized TAG as an alternative carbon source when glucose became unavailable. In Figures 20B-20C, tracking the carbon from cells cultured with [U-13C] glucose and natural stearic acid indicated that β-oxidation could be a source of acetyl-CoA for carotenoid synthesis. The average and standard deviation of three independent experiments were shown. [Figure 20C]Figures 20B-20C, carbon tracing from cells cultured with [U-13C] glucose and natural stearic acid, demonstrated that β-oxidation could be a source of acetyl-CoA for carotenoid synthesis. Means and standard deviations of three independent experiments are shown.

[0034] [Figure 21A] Bioreactor fermentation of β-carotene and lycopene engineered strains. Figures 21A and 21D, Fermentation profiles of β-carotene producing strain YLMA15 (Figure 21A) and lycopene producing strain YLMA34 (Figure 21D) in 3-L bioreactors. [Figures 21B-21D] Figures 21A and 21D, Fermentation profiles of β-carotene producing strain YLMA15 (Figure 21A) and lycopene producing strain YLMA34 (Figure 21D) in 3-L bioreactors. Figures 21B and 21E, β-carotene cultures were deep red-orange after 240 h of cultivation (Figure 21B), and lycopene cultures were deep red (Figure 21E). Figures 21C and 21F, Microscopic images of cells producing β-carotene (Figure 21C) and lycopene (Figure 21F). β-carotene and lycopene were shown to accumulate intracellularly and distributed throughout the cytoplasm in most cells. For Figures 21A and 21D, the average and standard deviation of three independent experiments are shown. [Fig. 21E-21F] Figures 21B and 21E, β-carotene cultures were deep red-orange after 240 hours of culture (Figure 21B), and lycopene cultures were deep red (Figure 21E). Figures 21C and 21F, Microscopic images of cells producing β-carotene (Figure 21C) and lycopene (Figure 21F). β-carotene and lycopene were shown to accumulate intracellularly and to be distributed throughout the cytoplasm in most cells.

[0035] [Figure 22]Scheme of metabolic pathways leading to the production of β-carotene in Y. lipolytica. The engineered β-carotene biosynthetic pathway involved directly upregulated genes of the mevalonate pathway (black), isopentenol utilization pathway (IUP, white), and β-carotene synthesis (dotted line). HMG-CoA, hydroxymethylglutaryl-CoA; MVA, mevalonate; MVAP, mevalonate-5-phosphate; IP, isopentenyl monophosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; tHMGR, truncated HMG-CoA reductase; ERG12, mevalonate kinase; IDI, isopentenyl diphosphate isomerase; ERG20, geranyl / farnesyl diphosphate synthase; GGPPS, GGPP synthase. CrtYB or CarRP, bifunctional phytoene synthase / lycopene β-cyclase; CrtI or CarB, phytoene dehydrogenase; CK, choline kinase; IPK, isopentenyl phosphate kinase.

[0036] [Figure 23] Crispr-Cas9-mediated disruption of TRP1 in Y. lipolytica strain po1f. A single adenine deletion (single underline at position -110) caused a frameshift mutation (double underline) that abolished TRP1 activity.

[0037] [Figure 24A] Effect of engineering the lycopene β-cyclase step on β-carotene production. Figure 24A, CarRP was a bifunctional enzyme in which the R and P domains conferred lycopene cyclase and phytoene synthase activities, respectively. Two methods were used to isolate the cyclase activity: truncation after the R domain of the CarRP gene and a loss-of-function mutation (D409G) in the P domain. [Fig. 24B-24C]Figure 24B, Increasing the copy number of lycopene β-cyclase did not improve β-carotene synthesis. Figure 24C, Relative gene expression levels associated with increasing CarRP copy number. [Figure 24D] Figure 24D, Expressing lycopene β-cyclase from various other organisms led to improved β-carotene titers. However, the increase was small and accumulation of lycopene was still observed. The mean and standard deviation (sd) of three independent experiments are shown.

[0038] [Diagram 25] Levels of intermediates involved in the β-carotene synthesis pathway. Intracellular concentrations of biosynthetic intermediates from FPP to β-carotene were measured, with lycopene being the only aggregate precursor. Data shown are the mean and standard deviation of three independent experiments. [Figure 26] Structural prediction of lycopene β-cyclase in CarRP. Schematic diagram of the CarRP protein showing the six transmembrane helices within the R domain (β-cyclase). [Figure 27] Computational model of the R domain of CarRP (lycopene cyclase). The TrRosetta confidence score of this model was 0.79, suggesting high certainty of the structural model.

[0039] [Figure 28] Clustering of variants to show distance between sequences. Variants were clustered using PhyML to ensure the spread of tested variants. [Figure 29] Relative gene expression levels associated with CarRP variants. The relative expression levels of gene CarRP in engineered strains harboring mutant CarRP and wild-type CarRP (WT) were quantified by RT-PCR. ACT1 was used as an internal control gene for normalization. Data shown were the mean and standard deviation of three independent experiments.

[0040] [Diagram 30]Spatial mapping of substitutions that remove substrate inhibition. The positions of successful variants were mapped onto a computational model of lycopene cyclase, with Y27R, V175W, and T31R-F92W shown as spheres. All substitutions appear to be located in the same spatial region of the enzyme.

[0041] [Diagram 31] Effects of isoprenol or prenol on cell growth. Isopentenol isomers isoprenol or prenol were fed at various concentrations to the po1f strain in YPD medium. OD600 was measured after 24 h of cultivation. Based on these results, 30 mM isoprenol or 10 mM prenol proved to be suitable concentrations for all subsequent experiments. Data shown were the mean and standard deviation of three independent experiments.

[0042] [Figure 32A] Disruption of lycopene cyclase activity in CarRP. Figure 32A, The E78K mutation in the R domain of CarRP results in loss of function of the cyclase activity, as indicated by the dotted rectangle. [Figure 32B] FIG. 32B, HPLC chromatograph showed that mutated CarRPE78K completely abolished β-carotene formation.

[0043] [Diagram 33] Manipulation of the MVA pathway and IUP further promoted lycopene biosynthesis. Overexpression of native genes in the MVA pathway improved lycopene synthesis. Furthermore, additional introduction of IUP into the lycopene-producing strain further enhanced the titer. Data shown were the mean and standard deviation of three independent experiments. [Diagram 34] Composition of modified YPD and YNB media used in this study.

[0044] [Diagram 35]Determination of the optimal initial glucose concentration for β-carotene production. Cells were cultured in Y5P10Dn medium (where n represents the initial glucose concentration). After 3 days of fermentation, the amount of glucose consumed, OD600, and β-carotene titer were measured. The optimal initial glucose concentration was found to be 50 g / L. Above that, the strain performance was adversely affected and the glucose consumption rate also decreased, probably due to osmotic stress. The mean and standard deviation of three independent experiments are shown. Statistical differences were analyzed using Student's t-test, and P<0.05 was considered statistically significant. *P<0.05.

[0045] [Diagram 36] Comparison of cell growth in media with various C / N ratios. Strain YLMA15 was used in these experiments, and biomass was found to decrease with increasing C / N ratio. Data shown are the mean and standard deviation of three independent experiments. [Figure 37] The optimized Y10P10D50 medium was used to further enhance the lycopene titer. Data shown were the mean and standard deviation of three independent experiments.

[0046] [Figure 38] Micromorphology of YLMA15 cells during fermentation. Cells collected at different time points during the culture were visualized by microscopy, and the observations were consistent with the fermentation profile. In the presence of glucose in the medium during the first 3 days, intracellular lipid droplets gradually aggregated into lipid bodies, which sequestered the produced β-carotene. However, due to the degradation of TAG, lipid bodies became invisible at later stages of glucose depletion, resulting in the accumulated β-carotene being better dispersed throughout the cells.

[0047] [Figure 39] HPLC chromatograph of carotenoids from YLMA15 after fed-batch fermentation. The selectivity for β-carotene was calculated based on the relative contents of lycopene and β-carotene. [Fig. 40A-40B]Correlation between dry cell weight (DCW) and OD600 in β-carotene-producing cells (FIG. 40A) and in lycopene-producing cells (FIG. 40B). DCW was calculated based on the measured OD600 by applying a conversion factor.

[0048] Detailed Description The present disclosure relates to a method for converting industrial wastes (e.g., dairy wastes) into valuable food and feed ingredients (e.g., carotenoids) and / or microbial feeds using engineered yeast cells. These ingredients can be naturally occurring products that belong to the family of isoprenoids (also called terpenoids) and are mainly synthesized by plants. Specifically, the present disclosure describes the synthesis of carotenoid compounds such as lycopene, β-carotene, and astaxanthin from dairy industry wastes, as well as metabolic and protein engineering strategies for their enhanced synthesis. Other products of the isoprenoid family can be synthesized from acid whey (AW) wastes as well.

[0049] Currently, the most common uses of AW are either added directly to soil as fertilizer or mixed with silage to feed livestock. In either case, however, the amount of AW that can be used is limited, and these are low-value uses. Conversely, processing AW in wastewater treatment plants increases the production costs of many foods. Overall, no adequate solution exists if large amounts of potential fermentable nutrients such as lactose, galactose, and lactic acid remain untapped (Menchik, et al. 2019). Moreover, this yellowish AW by-product is not attractive to the food industry because its sour and salty taste, high levels of ash, and low levels of protein limit its food applications (Lievore, et al. 2015).

[0050] Companies in the dairy industry have attempted to develop alternative approaches to process AW. As examples, Chobani (Norwich, NY) and Commonwealth Dairy (Brattleboro, VT) use reverse osmosis filtration systems to recover water from AW, reducing transportation costs. Others, such as General Mills (Minneapolis, MN), use anaerobic digestion to convert AW to methane, which can then be used to cover a portion of the plant's energy needs via methane-fed generators. However, these are all low-value-added processes, and the revenue generated from converting AW to methane has been relatively low. General Mills has also developed a method to neutralize AW for use in foods as a bulking agent or fortifier (US Patent Pub. 2014 / 0348981), or to produce oligosaccharides that can function as soluble fiber in cereals or baked goods (US Patent Pub. 2014 / 0348979). Danone describes a method to produce AW with a stable lactose content to allow for a more robust method of isolating lactose (International Patent No. WO 2016 / 177701 A1). Arla Food Ingredients (Viby, Denmark) and Ultima Foods (Quebec, Canada) are attempting to minimize AW production during food manufacturing using protein solutions and ultrafiltration, respectively.

[0051] Besides dairy companies, many research groups are exploring the use of AW. As an illustration, a method to produce glucose / galactose syrup and whey proteins from AW has been proposed using a combination of ultrafiltration and acid-catalyzed thermal hydrolysis of lactose (Lindsay et al., 2018). However, this approach requires expensive ultrafiltration membranes to isolate whey proteins and high temperatures to hydrolyze lactose. Furthermore, undesirable acid-catalyzed degradation reactions limit product yields, resulting in low value of the final product. Medium-chain carboxylic acids (MCCAs), such as n-caproic acid, are another group of compounds that have been targeted as products of AW fermentation using microbiomes (Xu et al., 2018). The use of a single bioreactor resulted in low specificity for the production of MCCAs, necessitating the use of more complex systems that introduce the microbiome to different operating conditions in a stepwise manner. This system, which employs bioreactors in series, increases process costs and suffers from scalability challenges. Recently, AW was used as an alternative growth medium for microalgae for the production of the enzyme β-galactosidase (Bentahar et al., 2019). In another study, researchers attempted to convert lactose present in AW to galactooligosaccharides (GOS) using two commercially available β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis, but the GOS yield was low.

[0052] Furthermore, some reports mention the use of AW in fermented dairy beverages to take advantage of the nutrients and water replacement found in AW (Lievore et al., 2015; Skryplonek, et al., 2019). However, the salty and sour taste of AW impairs the desired traits of fermented beverages in terms of flavor, aroma, and aftertaste. In addition, it has been reported that the use of AW in fermented dairy products alters the viscosity and therefore the texture of the product, decreasing the preference in sensory evaluation (Lievore et al., 2015). Additional ingredients need to be added to mask the flavor and odor of AW, which increases the cost.

[0053] Natural products are a rich source of bioactive molecules whose diverse properties have underpinned many applications in the pharmaceutical, food, and flavor / fragrance industries (Atanasov et al., 2015; Cragg, 1998; Dhingra et al., 1999; Dzubak et al., 2006; Zhou et al., 2009). Due to their structural complexity and very low abundance in natural sources, chemical synthesis and extraction of these compounds from plants has been particularly challenging (Chemler and Koffas, 2008; Martin et al., 2003), prompting efforts in their production by engineered microorganisms. Most metabolic engineering efforts for chemical manufacturing have focused primarily on the manipulation of functional reconstitution of metabolic pathways in the cytosol. However, this strategy often results in reduced yields or the formation of unwanted by-products due to extensive crosstalk and complex cellular metabolism involving elaborate regulatory mechanisms (Ajikumar et al., 2010; Martin et al., 2003). In this regard, the natural subcellular compartmentalization of eukaryotic cells can inspire a type of metabolic engineering that can successfully address these challenges (Hammer and Avalos, 2017).

[0054] The present disclosure also relates to the construction of engineered yeast cells by applying metabolic and protein engineering strategies that allow for the production of intracellular carotenoid compounds at high concentrations using either glucose or AW as feedstock. After purification steps, the carotenoid compounds can be used as antioxidants (food fortification), food colorings, dietary supplements, feed additives, and in cosmetic or personal care products. Another product can be a microbial feed enriched with carotenoids.

[0055] The main advantages of this technology are: (1) no pretreatment of AW is required and the bioprocess can be carried out under non-sterile conditions; (2) full utilization of AW to produce a water stream free of organic compounds; (3) improved process economics through synthesis of high-value specialty ingredients and co-production of microbial feed; (4) use of both dilute and concentrated AW; (5) simple and scalable fermentation process; (6) GRAS host microorganisms allow safe implementation of the technology in existing dairy production facilities with immediate access to ingredients; (7) footprint equivalent to that of an AW storage tank; and (8) significant increase in revenue compared to food production and reduction in costs associated with waste disposal and transportation of AW to farms, increasing revenue by up to 38% compared to current Greek yogurt production, for example.

[0056] The present disclosure also relates to a method for compartmentalizing metabolic pathways within yeast subcellular organelles. Subcellular organelles have attracted increasing attention due to their unique physicochemical environment and content of enzymes, metabolites, and cofactors that may provide favorable conditions for the functioning of various metabolic pathways (Ayer et al., 2013; Hammer and Avalos, 2017). Assembling pathways within smaller subcellular compartments can increase local substrate and enzyme concentrations, not only increasing reaction rates but also preventing the diversion of intermediates to competing pathways (Avalos et al., 2013). To date, most investigations of metabolic pathway compartmentalization in yeast have been performed in the model organism Saccharomyces cerevisiae, including: utilizing mitochondria for fuels, chemicals, and drugs (Avalos et al., 2013; Farhi et al., 2011; Szczebara et al., 2003; Yuan and Ching, 2016), compartmentalizing pathways in peroxisomes (Sheng et al., 2016; Zhou et al., 2016), targeting the ER and Golgi apparatus for fuel and drug production (Thodey et al., 2014), and vacuolar compartmentalization (Bayer et al., 2009). Targeting biosynthetic pathways to these subcellular compartments aids in the production of products of interest in this organism. Meanwhile, despite these advantages of organelle engineering, its potential in the oleaginous yeast Yarrowia lipolytica remains largely untapped. Because this yeast has the unique ability to accumulate large amounts of intracellular lipids in the form of lipid droplets, an opportunity exists to exploit the potential of pathway compartmentalization.

[0057] Astaxanthin is a high-value carotenoid derivative pigment that has attracted increasing interest due to its wide range of applications in food, animal feed, nutraceutical, cosmetic, and pharmaceutical industries (Ambati et al., 2014). These applications are due to its strong antioxidant activity (Hama et al., 2012), anti-inflammatory activity (Bennedsen et al., 2000), and anticancer activity (Chew et al., 1999). Traditional methods for the production of astaxanthin include chemical synthesis and extraction from natural sources. However, biosafety concerns from chemical routes and the high cost and variability of products produced by extraction routes limit its widespread application (Qi et al., 2020). Alternatively, metabolic pathway engineering for astaxanthin biosynthesis has been successfully demonstrated in various host organisms, generally occurring within the cytoplasm of the cell (Diao et al., 2020; Gong et al., 2020; Henke et al., 2018; Jiang et al., 2020; Jin et al., 2018; Kildegaard et al., 2017; Lemuth et al., 2011; Li et al., 2020; Lu et al., 2017; Ma et al., 2016; Nogueira et al., 2019; Park et al., 2018; Qi et al., 2020; Scaife et al., 2009; Scaife et al., 2012; Tramontin et al., 2019; Ukibe et al., 2009; Wang et al., 2017; Zhang et al., 2018; Zhou et al., 2019; Zhou et al., 2017; Zhou et al., 2015). The astaxanthin yields in these engineered microorganisms are rather low for cost-effective commercialization, mainly due to the low conversion efficiency of the precursor β-carotene to astaxanthin.

[0058] Metabolic engineering approaches to produce high-value chemicals in microorganisms mainly use the cytosol as a common reaction vessel. However, efficient synthesis of target compounds in the cytosol is often hindered by enzyme and substrate sequestration, as well as metabolic crosstalk. Compartmentalization of organelles in eukaryotic cells suggests a way to overcome these challenges. In some embodiments, the present disclosure relates to the expression of the astaxanthin biosynthetic pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. In some embodiments, the enzymes of the astaxanthin pathway can be fused to each other to improve substrate activity and reaction efficiency. In some embodiments, the fusion of two enzymes that convert β-carotene to astaxanthin, β-carotene ketolase and hydroxylase, works better than the expression of the individual enzymes. In some embodiments, the individual or fused enzymes of the astaxanthin biosynthetic pathway are expressed in the fat body, endoplasmic reticulum, or peroxisomal compartments. In some embodiments, targeting the astaxanthin pathway to intracellular organelles not only accelerates the conversion of β-carotene to astaxanthin, but also significantly reduces the accumulation of ketocarotenoid intermediates.

[0059] The present disclosure relates, at least in part, to methods and compositions for producing carotenoids from acid whey. Aspects of the present disclosure relate to modified yeast cells (e.g., oleaginous yeast cells) capable of producing carotenoids from acid whey. The term "oleaginous yeast cells" as used herein refers to yeast cells that are rich in membrane structures and intracellular compartments, which provide an ideal hydrophobic environment for metabolic engineering and the manufacture of industrial products. In some embodiments, the oleaginous yeast cells are oleaginous yeast cells that utilize acetate for cell growth and product synthesis. For example, in some embodiments, the oleaginous yeast cells are Yarrowia lipolytica cells. Y. lipolytica is a non-pathogenic oleaginous yeast that can utilize a variety of carbon sources, including organic acids, hydrocarbons, and various fats and oils. The term "oleaginous" refers to a microorganism that can accumulate more than 20% of its dry cell weight as lipids (see C. Ratledge et al., Microbial routes to lipids. Biochem Soc Trans. 1989 December; 17(6):1139-41). Exemplary oleaginous cells include, for example, yeasts such as Yarrowia lipolytica, Candida 107, Rhodotorula glutinis, Rhodosporidium toruloides, Cryptococcus curvatus, Trichosporon pullulan, Lipomyces lipofer, Schwanniomyces occidentalis and other species such as Yarrowia, Lipomyces, Rhodosporidium, and Cryptococcus; oleaginous bacteria such as Rhodococcus, Acinetobacter, and Streptomyces; and oleaginous algae and microalgae.

[0060] Aspects of the present disclosure relate to genetically engineered yeast cells (modified cells), including: a heterologous gene, where the heterologous gene encodes an enzyme having β-galactosidase (LacA) activity; one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate; one or more heterologous genes encoding one or more enzymes of the Leloir pathway; and one or more heterologous genes encoding one or more enzymes of the mevalonate pathway. In some embodiments, the modified cell is an oleaginous yeast cell. In some embodiments, the oleaginous cell is a Yarrowia lipolytica cell. In some embodiments, a polynucleotide comprising a gene is delivered to the cell. In some embodiments, the cell comprises a polynucleotide comprising a gene. In some embodiments, an enzyme encoded by the gene is delivered to the cell. In some embodiments, the cell comprises an enzyme encoded by the gene. In some embodiments, the gene is a heterologous gene. In some embodiments, the polynucleotide is a heterologous polynucleotide. In some embodiments, the enzyme is a heterologous enzyme. As used herein, the term "heterologous" is used interchangeably with the terms "recombinant" and "exogenous." A heterologous gene, polynucleotide, or enzyme refers to a gene, polynucleotide, or enzyme introduced or expressed into a host cell. A heterologous gene is a gene introduced or expressed into a host cell. A heterologous polynucleotide is a polynucleotide introduced or expressed into a host cell. A heterologous enzyme is an enzyme introduced or expressed into a host cell. In some embodiments, a heterologous gene, polynucleotide, or enzyme is from a different organism or species than the host cell. In some embodiments, a heterologous gene, polynucleotide, or enzyme is a synthetic gene, polynucleotide, or enzyme. In some embodiments, a heterologous gene, polynucleotide, or enzyme is an additional copy of a gene, polynucleotide, or enzyme endogenously expressed by a host cell. In some embodiments, a heterologous gene can be modified by mutation. The term "mutation" as used herein refers to a change, alteration, or modification of a nucleotide in a nucleic acid compared to its wild-type sequence.For example, without limitation, the mutation may include substitution, insertion, deletion, or any combination thereof. In some embodiments, at least one mutation is present. In some embodiments, more than one mutation is present. In some embodiments, when more than one mutation is present, the mutations are distinct (e.g., not of the same type (e.g., substitution, insertion, deletion)). In some embodiments, when more than one mutation is present, the mutations are identical (e.g., not of the same type (e.g., substitution, insertion, deletion)). In addition, in some embodiments, the mutation results in a frameshift.

[0061] A mutation, as described herein, is a region (e.g., a segment, part, nucleobase, nucleoside, nucleotide) of a given nucleic acid (e.g., DNA, RNA) that is different compared to the wild-type nucleic acid, and will in most cases be reflected in each strand of the nucleic acid. That is, if a mutation is present in a sample, the mutation and its complement will be observed in each strand of the nucleic acid when sequenced. However, this is problematic given that a sample may contain single-stranded portions (e.g., gaps, overhangs), or regions that may induce strand resynthesis (e.g., nicks). This problem occurs because if a damaged base is present in such a single-stranded region, or in another region that is resynthesized, the damaged base may direct the synthesis of its complementary strand to include a base that was not originally present in the nucleic acid from which the sample was generated (because the damaged base may affect non-standard base pairs). The same can happen if one strand contains a mismatched base. In such cases, the mismatch will be displayed as a pair match of the resynthesized complement, rather than its original mismatched base. When this occurs, sequencing of both strands will read the mutations in each strand, thus indicating a mutation, but this mutation may not truly reflect the original nucleic acid. Such mutations are referred to herein as "pseudomutations." Pseudomutations are mutations that result from the resynthesis of a complementary strand of a nucleic acid, and do not represent the complementary strand of the original (e.g., natural, wild-type) nucleic acid from which the sample was obtained.

[0062] The terms "wild-type" and "native", which may be used interchangeably herein, are terms understood by those of skill in the art and refer to the typical form of an item, organism, strain, gene, or characteristic that occurs in nature and is distinct from engineered, mutated, or variant forms.

[0063] In some embodiments, the one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate are selected from the group consisting of lactate transporter (JEN1) and lactate dehydrogenase (LDH). In some embodiments, the one or more heterologous genes encoding one or more enzymes of the Leloir pathway are selected from the group consisting of GAL10M, GAL1, GAL7, and GAL10E.

[0064] The Leloir pathway is a metabolic pathway known in the art. The Leloir pathway is used for the catabolism of D-galactose by cells. The term "catabolism" as used herein refers to the metabolic process of breaking down complex molecules (e.g., D-galactose) in living organisms to form simpler molecules (e.g., glucose-1-phosphate). As known to those skilled in the art, the Leloir pathway converts galactose into glucose-1-phosphate through the enzyme activities of GAL10M, GAL1, GAL7, and GALE, among others. In some embodiments, the Leloir pathway is used to produce pyruvate in modified cells.

[0065] In some embodiments, the one or more heterologous genes encoding one or more enzymes of the mevalonate pathway are selected from the group consisting of geranylgeranyl diphosphate synthase (GGPPS), phytoene synthase (CarRP), and phytoene desaturase (CarB). In some embodiments, the GGPPS is GGPPSxd from Xanthophyllomyces dendrorhous, GGPPSsa from Sulfolobus acidocaldarius, GGPPStc from Taxus canadensis, GPPSpa from Pantoea agglomerans, GGPPSyl from Yarrowia lipolytica.

[0066] The mevalonate (MVA) pathway is another metabolic pathway known in the art. The MVA pathway is also known as the isoprenoid pathway or the HMG-CoA reductase pathway, and is an important metabolic pathway that produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAP) from acetyl-CoA. In some embodiments, the IPP and DMAP in the MVA pathway are further metabolized to farnesyl diphosphate (FPP). In some embodiments, the FPP in the MVA pathway is further metabolized to geranylgeranyl pyrophosphate (GGPP) by GGPPs (e.g., GGPPSxd, GGPPSsa, GGPPStc, GGPPSpa, GGPPSyl). In some embodiments, the GGPP in the MVA pathway is further metabolized to phytoene by CarRP. In some embodiments, the phytoene in the MVA pathway is further metabolized to lycopene by CarB.

[0067] In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having lycopene beta cyclase activity. The amino acid sequence of the lycopene beta cyclase enzyme is provided herein as SEQ ID NO:1: MLLTYMEVHLYYTLPVLGVLSWLSRPYYTATDALKFKFLTLVAFTTASAWDNYIVYHKAWSYCPTCVTAVIGYVPLEEYMFFIIMTLLTVAFTNLVMRWHLHSFFIRPETPVMQSVLVRLVPITALLITAYKAWHLAVPGKPLFYGSCILWYACPVLALLWFGAGEYMMRRPLAVLVSIALPTLFLCWVDVVAIGAGTWDISLATSTGKFVVPHLPVEEFMFFALINTVLVFGTCAI (SEQ ID NO: 1)

[0068] In some embodiments, the enzyme having lycopene β-cyclase activity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 1. The terms "percent identity", "sequence identity", "% identity", "% sequence identity" and "% identical" as used interchangeably herein refer to a quantitative measure of similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequences, intron and exon sequences, and amino acid sequences between humans and other species varies by species type, with chimpanzees having the highest percent identity with humans among all species in each category.

[0069] The calculation of the percent identity of two nucleic acid sequences can be carried out, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps to be introduced for optimal alignment of the two sequences and the length of each gap.

[0070] Comparison of sequences and determination of identity percentage between two sequences can be carried out using mathematical algorithm.For example, the identity percentage between two nucleotide sequences can be determined using the method described in: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software for determining the homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA Atschul, SF et al., J. Molec. Biol., 215, 403 (1990).

[0071] When a percent identity or range (e.g., at least, more, etc.) is recited, the endpoints are inclusive unless otherwise specified and a range (e.g., at least 70% identity) is intended to include all ranges within the recited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 122%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 200%, at least 201%, at least 202%, at least 203, at least 204, at least 205, at least 206, at least 207, at least 208, at least 209, at "identity" is intended to include at least 5%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity) and all increments thereof (e.g., tenths of a percent (i.e., 0.1%), hundredths of a percent (i.e., 0.01%), etc.).

[0072] In some embodiments, the lycopene beta cyclase enzyme (SEQ ID NO: 1) is modified or mutated to increase or decrease enzymatic activity. In some embodiments, the enzyme having lycopene beta cyclase activity comprises an amino acid sequence represented by any one of SEQ ID NOs: 2-4. In some embodiments, the enzyme comprises the following amino acid substitutions relative to SEQ ID NO: 1: Y27R; V175W; T31R; F92W; or T31R and F92W.

[0073] The amino acid sequence of one such modified lycopene β-cyclase activity is provided herein as SEQ ID NO:2: MLLTYMEVHLYYTLPVLGVLSWLSRPRYTATDALKFKFLTLVAFTTASAWDNYIVYHKAWSYCPTCVTAVIGYVPLEEYMFFIIMTLLTVAFTNLVMRWHLHSFFIRPETPVMQSVLVRLVPITALLITAYKAWHLAVPGKPLFYGSCILWYACPVLALLWFGAGEYMMRRPLAVLVSIALPTLFLCWVDVVAIGAGTWDISLATSTGKFVVPHLPVEEFMFFALINTVLVFGTCAI (SEQ ID NO: 2)

[0074] The amino acid sequence of another such modified lycopene β-cyclase activity is provided herein as SEQ ID NO:3: MLLTYMEVHLYYTLPVLGVLSWLSRPYYTATDALKFKFLTLVAFTTASAWDNYIVYHKAWSYCPTCVTAVIGYVPLEEYMFFIIMTLLTVAFTNLVMRWHLHSFFIRPETPVMQSVLVRLVPITALLITAYKAWHLAVPGKPLFYGSCILWYACPVLALLWFGAGEYMMRRPLAWLVSIALPTLFLCWVDVVAIGAGTWDISLATSTGKFVVPHLPVEEFMFFALINTVLVFGTCAI (SEQ ID NO: 3)

[0075] The amino acid sequence of yet another such modified lycopene β-cyclase activity is provided herein as SEQ ID NO:4: MLLTYMEVHLYYTLPVLGVLSWLSRPYYTARDALKFKFLTLVAFTTASAWDNYIVYHKAWSYCPTCVTAVIGYVPLEEYMFFIIMTLLTVAWTNLVMRWHLHSFFIRPETPVMQSVLVRLVPITALLITAYKAWHLAVPGKPLFYGSCILWYACPVLALLWFGAGEYMMRRPLAVLVSIALPTLFLCWVDVVAIGAGTWDISLATSTGKFVVPHLPVEEFMFFALINTVLVFGTCAI (SEQ ID NO: 4)

[0076] In some embodiments, the modified cell further comprises a heterologous gene encoding tHMGR, ERG12, IDI, and ERG20 of the mevalonate (MVA) pathway, and / or choline kinase (CK) and isopentenyl phosphate kinase (IPK). In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having β-carotene ketolase (CrtW) activity; and a heterologous gene encoding an enzyme having β-carotene hydroxylase (CrtZ) activity. In some embodiments, the enzyme having CrtW activity is fused to an enzyme having CrtZ activity. In some embodiments, the CrtW / CrtZ fusion enzyme comprises a localization signal. In some embodiments, the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisome, and / or fat body. The term "fusion enzyme" as used herein refers to an enzyme protein that comprises two or more separate proteins. In some embodiments, the fusion enzyme is made through the joining of two or more genes that originally encode separate proteins. In some embodiments, two or more genes linked together are translated into a single protein or enzyme. The term "localization signal" refers to a peptide fragment expressed on a protein of interest that mediates the transport of said protein to a target location inside or outside the cell. In some embodiments, the localization signal is a short peptide fragment. In some embodiments, the localization signal targets the protein to the endoplasmic reticulum. In some embodiments, the localization signal targets the protein to the peroxisome. In some embodiments, the localization signal targets the protein to the fat body of the cell.

[0077] In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having lycopene β-cyclase activity and / or a heterologous gene encoding an enzyme having lycopene ε-cyclase activity.In some embodiments, the modified cell further comprises a heterologous gene encoding an enzyme having carotenoid hydroxylase 1 (LUT1) activity and / or a heterologous gene encoding an enzyme having carotenoid hydroxylase 5 (LUT5) activity.

[0078] Another aspect of the present disclosure relates to a genetically engineered yeast cell (modified cell) comprising: a first heterologous gene, where the first heterologous gene encodes an enzyme having β-carotene ketolase (CrtW) activity; and a second heterologous gene, where the second heterologous gene encodes an enzyme having β-carotene hydroxylase (CrtZ) activity; where the modified cell produces β-carotene. In some embodiments, the modified cell is an oleaginous yeast cell. In some embodiments, the oleaginous cell is a Yarrowia lipolytica cell. In some embodiments, the enzyme having CrtW activity is fused to an enzyme having CrtZ activity. In some embodiments, the CrtW / CrtZ fusion enzyme comprises a localization signal. In some embodiments, the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisome, and / or fat body.

[0079] Another aspect of the present disclosure relates to a method of converting a carbon source to lycopene and / or β-carotene, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient time to convert the carbon source to lycopene and / or β-carotene. In some embodiments, the carbon source is acid whey. In some embodiments, the carbon source is converted to lycopene. In some embodiments, the carbon source is converted to β-carotene. As used herein, the term "carbon source" refers to any natural or artificial source of carbon, such as carbon dioxide, methane, or acid whey.

[0080] Another aspect of the present disclosure relates to a method of converting a carbon source to astaxanthin, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient period of time to convert the carbon source to astaxanthin. In some embodiments, the carbon source is acid whey. Another aspect of the present disclosure relates to a method of converting a carbon source to alpha-carotene, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient time to convert the carbon source to alpha-carotene. In some embodiments, the carbon source is acid whey.

[0081] Another aspect of the present disclosure relates to a method of converting a carbon source to lutein, comprising: contacting the modified cells described herein with a carbon source; and incubating the modified cells with the carbon source for a sufficient period of time to convert the carbon source to lutein. In some embodiments, the carbon source is acid whey. Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 2. Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 3. Another aspect of the present disclosure relates to an enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 4.

[0082] Details of one or more embodiments of the methods and products disclosed herein are set forth in the following description. Other features or advantages of the methods and products disclosed herein will be apparent from the following drawings and detailed description of some embodiments, as well as the appended claims. The term "approximately" or "about" may be used interchangeably herein and applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (i.e., percentage greater or less) of the stated reference value, unless otherwise stated or clear from the context (e.g., when such number exceeds 100% of possible values).

[0083] The phraseology and terminology used in this application is for the purpose of description and should not be regarded as limiting. The use of "including," "including," "having," "containing," "involving," and variations thereof in this application is meant to encompass the items listed thereafter and equivalents thereof, as well as additional items. The methods and products disclosed herein are further illustrated by the following examples, which should not be construed as further limiting in any way. The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. EXAMPLES

[0084] In order that the methods and products described herein may be more fully understood, the following examples are set forth. The examples described herein are presented to illustrate the systems and methods provided in this disclosure and should not be construed as limiting the scope thereof in any manner.

[0085] Example 1. Engineering microorganisms to produce carotenoids from acid whey This example concerns the engineering of the oleaginous yeast Yarrowia lipolytica to overproduce carotenoids from acid whey and / or animal feed with oils and proteins of various compositions as required. Previously, it was shown that Y. lipolytica can consume all sugars in AW by expression of secreted extracellular β-galactosidase and expression of genes driving the native galactose metabolic pathway (Mano et al., 2020). Herein, an optimal gene combination for lycopene synthesis and two independent strategies to almost completely avoid substrate inhibition during β-carotene synthesis are described. Substrate inhibition was undesirable in industrial applications of microorganisms used for product synthesis. To address this limitation, several methods such as enzyme immobilization, two-phase partitioning bioreactor systems, batch substrate feeding strategies, and protein engineering were explored, but most of these solutions were limited to systems where inhibition was provided by the starting substrate and were difficult to apply in the context of microbial engineering for chemical production. It should be noted that the strategy was first demonstrated in a carotenoid-producing strain of Y. lipolytica that uses glucose as a carbon source, and the results subsequently informed the engineering of yeast cells for efficient production of carotenoid compounds from AW.

[0086] Biosynthesis of β-carotene and lycopene from glucose First, structure-guided protein design combined with phylogenetic information was used to generate protein variants with reduced inhibition. As an illustration, one mutation in the lycopene cyclase gene was identified, which completely abolished substrate inhibition without reducing enzyme activity and significantly increased β-carotene production. Synthesis of β-carotene in Y. lipolytica required heterologous expression of three genes encoding the enzymes phytoene synthase, phytoene dehydrogenase, and lycopene cyclase. Geranylgeranyl diphosphate synthase (GGPPS) was also considered, since it controlled the flux directed towards carotenoid synthesis instead of sterols. The relevant genes were sourced from eukaryotes and introduced into the po1f strain of Y. lipolytica, which carries a disruption of TRP1.

[0087] The second approach resulted in similar potency and selectivity for β-carotene by reducing carbon flow through the carotenoid pathway, preventing the accumulation of inhibitory metabolites to inhibitory levels, contrary to the traditional paradigm of pathway engineering. This was achieved by establishing a metabolic valve through geranylgeranyl pyrophosphate synthase (GGPPS) that regulates the rate of formation of the substrate lycopene, effectively alleviating substrate inhibition. Although this approach reduced flux through the pathway of interest, the benefits of maintaining high enzyme activity at suppressed substrate levels overcompensated for the loss of productivity due to flux diversion.

[0088] It was also noted that the product profile of β-carotene versus lycopene could be altered by altering GGPPS activity in vivo. High activity of GGPP could be exploited to reconstitute a dedicated lycopene-producing strain. Combining this idea with a specific lycopene cyclase variant, we successfully produced high concentrations of lycopene together with undetectable amounts of β-carotene. Further increase in lycopene production was achieved by overexpressing the MVA pathway and introducing IUP with glucose as substrate.

[0089] Previous studies have demonstrated that the lipophilic nature of carotenoids promotes their storage in the cellular lipid body. found that an engineered lipid-overproducing strain could produce more β-carotene at a titer of 6.5 g / L with the production of 42.6 g / L of lipids (Larroude et al., 2018). While providing a compatible compartment for hydrophobic isoprenoid accumulation, the de novo formation of TAG also consumes large amounts of carbon sources, limiting acetyl-CoA flux to the MVA and product formation pathways. This study demonstrated that the intrinsic TAG accumulation capacity of Y. lipolytica is sufficient for carotenoid sequestration. By balancing the flux distribution of carotenoid synthesis and lipid synthesis through tuning the C / N ratio, a large portion of the acetyl-CoA pool was reserved for carotenoid production, achieving higher titers and content per cell.

[0090] Furthermore, it was demonstrated that carotenoid biosynthesis during the glucose-depleted stationary phase was supported by cellular lipid degradation. During the fermentation experiments with glucose as a substrate, it was found that the content of β-carotene continued to rise even after glucose in the medium was depleted during the stationary phase. Throughout the fermentation process, the lipid content initially increased, reached a maximum on the third day, and then rapidly decreased when glucose was completely consumed. However, despite the decrease in lipid content, the β-carotene content continued to rise far beyond the point of glucose depletion, suggesting that TAG was utilized to maintain metabolic activity and carotenoid synthesis. When glucose was still present during the first three days, the lipid droplets in the cells gradually aggregated into lipid bodies, sequestering the generated β-carotene. However, the lipid bodies were no longer visible in the later stages of fermentation due to the degradation of TAG, and then the accumulated β-carotene was dispersed throughout the cells. It was also demonstrated that acetyl-CoA formed by β-oxidation supports the MVA pathway and ultimately contributes to β-carotene formation. Therefore, this is likely the mechanism used by cells to convert TAG to carotenoids during glucose-depleted stationary phase.

[0091] Biosynthesis of β-carotene and lycopene from AW After obtaining results for carotenoid synthesis using glucose as a substrate, we constructed a Y. lipolytica strain that could completely consume all organic molecules found in AW and produce high concentrations of lycopene. Engineering efforts included the introduction of genes involved in lactose degradation, galactose assimilation, highly active GGPP, phytoene dehydrogenase, and a bifunctional phytoene synthase / lycopene cyclase of eukaryotic origin with a single mutation that abolished cyclase activity. The flux distribution of lycopene synthesis and lipid synthesis was balanced by adjusting the C / N ratio, which achieved a maximum lycopene concentration of about 3 g / L with 0.230 mg lycopene per g dry cell weight using untreated AW as a substrate. The engineered strain was also capable of consuming concentrated AW. After 14 days, all sugars and organic acids in AW were completely consumed, resulting in a maximum lycopene concentration of 13.4 g / L.

[0092] Astaxanthin biosynthesis Another high-value carotenoid derivative pigment is astaxanthin, which has attracted increasing interest due to its wide range of applications in the food, animal feed, nutraceutical, cosmetic, and pharmaceutical industries due to its strong antioxidant, anti-inflammatory, and anti-cancer properties.

[0093] The downstream astaxanthin biosynthetic pathway from β-carotene was constructed by expressing the CrtW gene encoding β-carotene ketolase and the CrtZ gene encoding β-carotene hydroxylase. To further improve the heterologous pathway, β-carotene ketolases and hydroxylases from diverse organisms were procured. Given that the main natural sources of astaxanthin synthesis are bacteria and algae, additional CrtW and CrtZ were specifically screened from these organisms and a β-carotene overproducing strain was used to identify the CrtW / Z pair that maximizes microbial astaxanthin production. Furthermore, we determined that increasing the physical proximity between CrtW and CrtZ could minimize the substrate-enzyme distance and increase the reaction rate. To that end, an active fusion of the CrtW and CrtZ enzymes was created, aiming to enhance the contact between the product / precursor and the corresponding enzyme by creating a tighter microenvironment. The fusion enzymes had higher efficiency in producing astaxanthin compared to the individual enzymes. Finally, we investigated the expression of the fusion enzyme when targeted to various subcellular compartments. Initially, the fusion enzyme CrtW-Z was targeted to the fat body (LB) by conjugating it to an oleosin sequence, thus providing an alternative biological pathway for astaxanthin biosynthesis. The LB-targeted strain yielded significantly higher titers of astaxanthin compared to a control strain expressing the pathway in the cytosol. Targeting the fusion enzyme to other organelles, such as the endoplasmic reticulum and peroxisomes, further improved the astaxanthin titers from glucose, and even better results were obtained by targeting all three sub-organelles simultaneously.

[0094] Other products The results described herein demonstrate the technique to engineer the oleaginous yeast Y. lipolytica for the biosynthesis of a variety of high-value added products. This technique is also applicable to the production of other products from AW, such as lutein, carotene, and other members of the isoprenoid pathway. Thus, the present disclosure relates to a general method for upgrading dairy industry waste to a collection of high-value added products that are widely used as food feedstocks.

[0095] Production of animal feed from AW Protein production is a natural component of the yeast life cycle and can represent approximately 40% of the dry weight under certain conditions (Yamada et al., 2005). Lipid production, on the other hand, is dependent on specific nutritional cues, usually depending on the growth phase (Goncalves et al., 2014). Early in fermentation, most energy and carbon are utilized for growth and cell division, but as essential nutrients (especially nitrogen) begin to be depleted, cells stop dividing and instead start storing excess carbon in the form of lipids, which are sequestered in large intracellular droplets. Under certain conditions, these lipid bodies can represent more than two-thirds of the cell dry weight by the end of fermentation (Qiao et al., 2015). In the case of AW, the available nitrogen is mainly in the form of milk proteins, and by modifying Yarrowia's access to this nitrogen source, it is possible to produce products with a higher or lower proportion of their weight that is lipid.

[0096] The lipid to protein ratio was controlled by utilizing engineered strains of Yarrowia. Two strains of Yarrowia were used; W29 (non-engineered), which produces a mixture of lipid and cell mass, and an engineered strain named ACC-DGA. The ACC-DGA strain was engineered to produce more lipid by overexpression of native Yarrowia genes that code for enzymes involved in the biosynthesis of triacylglycerol (Tai et al., 2013; US patent application US20130143282A1). These strains also differed in their ability to consume proteins present in AW. ACC-DGA was deficient in the production of secreted proteases and therefore was unable to degrade milk proteins. Starting with these two strains, either high fat, high protein, or a mixture of the two products were produced.

[0097] Example 2. Converting dairy waste into food and feed ingredients To expand the AW utilization technology and transform it into a platform for producing valuable food and feed ingredients, an engineered strain of Y. lipolytica capable of producing lycopene as a model compound of natural products from AW was required. It was subsequently observed that lycopene could serve as a precursor for other carotenoids such as α-carotene, β-carotene, lutein, and astaxanthin (Figure 2). To date, attempts have been reported to improve the production of β-carotene and astaxanthin, mainly by using model microorganisms E. coli and S. cerevisiae. However, recombinant production of α-carotene and lutein in fungi had not been reported. Moreover, based on previous studies, it was shown that Y. lipolytica could consume all the sugars in AW by expression of secreted extracellular β-galactosidase and expression of genes promoting the native galactose metabolic pathway (Figures 1A-1C and Figure 3). Therefore, this AW-utilizing strain was an excellent starting point to be used as a chassis for subsequent engineering steps (Figure 3).

[0098] To achieve this goal, first, the optimal combination of carotenoid biosynthetic enzymes that maximizes carotenoid production and accumulation in Yarrowia cells will be determined. For further improvement, optimal enzyme colocalization in subcellular organelles such as the endoplasmic reticulum, peroxisomes, and fat bodies will be identified using various subcellular localization strategies. Enzymes localized in different subcellular compartments will have higher activity in converting substrates localized in the same compartment. Finally, the native mevalonate pathway will be engineered by overexpressing well-known rate-limiting enzymes to increase the supply of carotenoid precursors.

[0099] Reverse osmosis has been commonly applied to concentrate AW to small volumes to reduce the costs of waste treatment and transportation. High concentrations of lactate in concentrated AW can inhibit the growth of Y. lipolytica. Thus, methods to enhance the tolerance of Y. lipolytica to lactate by overexpressing enzymes involved in lactate consumption will be sought. This approach can be complemented by manipulating oxidative stress defense pathways based on previous studies in which lipid synthesis was improved in Y. lipolytica (Xu et al., 2017). All constructed strains will be validated in 10 L large-volume bioreactors. Another approach is to investigate the genome-wide response of Y. lipolytica to concentrated AW, which will help understand the genomic basis of resistance to concentrated AW. Transcriptional analysis will evaluate the genome-wide response and allow the identification of genes central to resistance conferring and potential mechanisms underlying the enhanced resistance of strains. Starting from the engineered strains described in the previous objectives, genome-wide evolutionary engineering strategies can be applied to isolate mutants with increased resistance. Strain-specific genetic and global gene expression differences of the mutants will be identified using multi-omics analyses (genomics and transcriptomics) and inform rational engineering of the host strain.

[0100] Furthermore, TEA will be used to evaluate the potential feasibility of the proposed bioprocess and identify process and economic bottlenecks and targets for further research and improvement. Evaluation of the overall value of the proposed technology will provide useful information to potential investors. Process modeling will be performed using a process simulator. Environmental assessment of the proposed bioprocess is another aspect that will be considered to identify and focus on environmentally significant bioprocess parameters (Heinzle et al., 1998).

[0101] Additional research will also include optimizing the fermentation process and purifying the carotenoid product. As an intracellular product, carotenoids are sequestered within the cells and need to be extracted and purified from the cell biomass. Extraction methods may first include a pretreatment step that aids in the breakdown of the cell walls. Then, carotenoids are traditionally extracted using organic solvents since they are lipophilic. This process may include washing steps, crystallization steps, and removal of solvent traces by vacuum drying. Challenges with carotenoid extraction include the sensitivity of carotenoids to excess heat, light, acid, and long extraction times. However, it is noted that carotenoid separation and purification can be performed according to established technologies that can be licensed and deployed in an AW-to-carotenoid integration scheme.

[0102] Example 3. Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica Engineering microbes for the overproduction of high-value natural products has primarily focused on the manipulation of metabolic pathways in the cell cytoplasm. Recently, there has been growing interest in directing metabolic pathways to yeast intracellular organelles to increase local substrate and enzyme concentrations and enhance the efficiency of compartmentalized pathways and end-product production (Cao et al., 2020; Hammer and Avalos, 2017). Compared to the traditional model yeast S. cerevisiae, systematic studies of cellular compartments for natural product biosynthesis in the oleaginous yeast Y. lipolytica have lagged behind. This is despite the important roles played by intracellular compartments such as LB, ER, and peroxisomes in this yeast. In this study, we assembled a heterologous metabolic pathway for astaxanthin synthesis using the fusion enzyme CrtW-Z and targeted expression in intracellular organelles of Y. lipolytica. This disclosure relates to bringing precursors for astaxanthin synthesis into close proximity with enzymes that catalyze pathway reactions. As Y. lipolytica is widely recognized as a model organism for the production of acetyl-CoA-derived compounds ( Abdel-Mawgoud et al., 2018 ), other products derived from the same precursors may also benefit from this study.

[0103] In the first experimental round, we exploited the lipophilicity of β-carotene, the main precursor for astaxanthin synthesis, and targeted the lipophilic compartment of the lipid body for the expression of the astaxanthin pathway. After successful implementation of this strategy, we next targeted the ER, the compartment for β-carotene synthesis, which further increased production. Finally, peroxisomes were also targeted for compartmentalization, as they also serve as a reservoir for lipophilic compounds. Targeting the astaxanthin pathway to all three compartments yielded the best results in terms of product accumulation, suggesting that placing the astaxanthin pathway in close proximity to the β-carotene precursor and providing a suitable medium for the storage of astaxanthin is important for promoting product accumulation in Y. lipolytica. Simultaneous targeting of the CrtW-Z-dependent pathway to LB, ER, and peroxisomes resulted in the highest astaxanthin titers reported to date in yeast. Thus, utilizing intracellular organelles could be a promising approach to further enhance isoprenoid biosynthesis due to the potential benefits of improving precursor supply and cofactor availability.

[0104] Overall, this disclosure successfully explored enzyme fusion and its compartmentalization in intracellular organelles to effectively direct substrates or intermediates to the final product. These approaches are among the first in the development of intracellular cell factories in yeast. Although hydrophobicity was investigated as a key property in engineering the astaxanthin pathway, other molecular features may also be explored in designing future applications of organelle engineering.

[0105] Screening for combinations of β-carotene ketolase and hydroxylase enzymes The biosynthetic pathway of astaxanthin has been extensively studied and well characterized. As shown in Figure 4, glucose is converted into the precursor isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) via the glycolysis pathway and the mevalonate (MVA) pathway. IPP and DMAPP are then condensed by GGPP synthase to form geranylgeranyl diphosphate (GGPP). GGPP is then converted into astaxanthin by heterologous enzymes, including the downstream pathway from GGPP to astaxanthin. Thus, the heterologous pathway to astaxanthin synthesis can be divided into two modules: an upstream module from GGPP to β-carotene and a downstream module from β-carotene to astaxanthin (Figure 4). In a previous study, a Y. lipolytica strain was engineered to enable the upstream pathway to overproduce β-carotene. This was achieved by introducing the genes of the β-carotene biosynthetic pathway, namely GGPP synthase (GGPPsa) from Sulfolobus acidocaldarius, the gene for the bifunctional enzyme phytoene synthase / lycopene β-cyclase (CarRP), and the gene for phytoene dehydrogenase (CarB) from Mucor circinelloides, into the po1f gene of Y. lipolytica. In this β-carotene producing strain, the auxotrophic marker was removed by the Cre-loxP system to allow the next round of genomic integration. Thus, a chassis host strain capable of overproducing β-carotene, the substrate for astaxanthin synthesis, was obtained.

[0106] The downstream astaxanthin biosynthesis pathway from β-carotene was constructed by expressing the CrtW gene encoding β-carotene ketolase and the CrtZ gene encoding β-carotene hydroxylase, which add two keto moieties and a hydroxyl, respectively, changing the colony color from yellow to red (Figure 5A). The titers of β-carotene obtained by heterologous expression of CrtZ / W were much higher compared to those of astaxanthin, suggesting that ketonization and hydroxylation of β-carotene are steps in astaxanthin synthesis (Kildegaard et al., 2017). As a first attempt to construct an astaxanthin pathway, CrtW (PsCrtW) from the marine bacterium Paracoccus sp. and CrtZ (PaCrtZ) from the enterobacterium Pantoea ananatis were introduced into the β-carotene-producing strain YL00. This resulted in 6.1 mg / L astaxanthin after 72 h of incubation (Figure 5B). The low yield was likely due to the unbalanced enzyme activities that led to the accumulation of intermediates. It has been recognized that bacterial CrtW and CrtZ can utilize not only β-carotene but also its ketone and hydroxylated products as substrates, resulting in diverse carotenoid intermediate profiles that may significantly affect astaxanthin yield and productivity (Chang et al., 2015; Choi et al., 2005; Wang et al., 2017). Therefore, due to substrate preference and unbalanced enzyme activities, an optimal combination of β-carotene ketolase and hydroxylase activities is important to promote astaxanthin accumulation.

[0107] To further improve the heterologous pathway, we sourced β-carotene ketolases and hydroxylases from diverse organisms. As the main natural sources of astaxanthin synthesis are bacteria and algae, we further screened two CrtW and two CrtZ specifically from such organisms (Table 1). To identify the CrtW / Z pair that maximizes microbial astaxanthin production, we combinatorially screened three CrtW and three CrtZ pairs in β-carotene overproducing strains. Astaxanthin titers ranging from 3.2 to 9.9 mg / L were obtained in various genetically engineered strains (Figure 5B), indicating that balancing enzyme activities is essential to reconstruct an efficient pathway for astaxanthin production. The optimal combination of CrtW / Z resulting in the highest astaxanthin production is from PsCrtW / HpCrtZ (Figure 5B). Notably, all strains harboring PsCrtW performed much better than the other two CrtW-harboring strains (Figure 5B). Furthermore, HpCrtZ, an enzyme derived from a eukaryote, was successfully expressed in Y. lipolytica for the first time, resulting in high levels of astaxanthin production (Figure 5B).

[0108] Astaxanthin production is enhanced by fusion enzyme Depending on the order in which the ketonization and hydroxylation reactions are performed in the biosynthetic process from β-carotene to astaxanthin, several intermediates were synthesized, forming multiple pathways from β-carotene to the final product (Figure 6A). Although the engineered strain YL02 harboring an optimized combination of CrtW and CrtZ improved astaxanthin production, higher levels could be possible by converting other accumulated intermediates, especially echinenone, canthaxanthin, and zeaxanthin (Figure 6B). The nature of the accumulated intermediates suggested that the conversion of canthaxanthin or zeaxanthin to astaxanthin was a bottleneck in the pathway. It was hypothesized that these bottlenecks were due to low metabolite concentrations in the vicinity of the CrtZ / CrtW enzymes and could therefore be eliminated by reducing the distance between the enzymes and their substrates. Increasing the physical proximity between CrtW and CrtZ could minimize the distance between the substrate and the enzymes and increase the reaction rate. To test this hypothesis, we created active fusions between the CrtW and CrtZ enzymes, aiming to enhance contacts between products / precursors and the corresponding enzymes by creating a tighter microenvironment.

[0109] β-carotene ketolase (PsCrtW) from Paracoccus sp. and β-carotene hydroxylase (HpCrtZ) from Haematococcus pluvialis were selected to create a fusion enzyme based on their high activity to produce more astaxanthin when expressed individually (Figure 5B). These two proteins were expressed as a translational fusion, separated by a linker spacer introduced to bring the two enzymes in close proximity and at the same time allow for interaction between the domains. The two constructs were investigated by expressing the fusion and its control, respectively, in the β-carotene overproducing strain YL00 (Figure 6C). We measured the levels of astaxanthin and intermediates in the genetically engineered strains, showing that both engineered strains expressing CrtZ-W or CrtW-Z enzyme fusions were able to increase the production of astaxanthin (Figure 6C and Figure 9), suggesting that both fusion enzymes function similarly but are more active than the corresponding individually expressed enzymes. Compared to the control strains harboring the individual enzymes CrtW+Z, the production of astaxanthin by the fusion enzyme strains was significantly improved, with an increase of 2.2-fold (up to 22.8 mg / L) in the fusion CrtZ-W strain and 2.8-fold (up to 28.9 mg / L) in the fusion CrtW-Z strain (Figure 6C). This supports the hypothesis that the significant improvement of the fusion enzyme strains is likely due to improved local proximity of the substrate-enzyme and their interactions. Interestingly, the strain expressing the CrtW-Z fusion showed improved performance over the strain with the CrtZ-W fusion enzyme (Figure 6C), suggesting that the catalytic activity may be higher when CrtW is located at the N-terminus of the fusion enzyme. It should be noted that the metabolite profile of the fusion enzyme strains was different from that of the individual enzymes. The accumulation of intermediates, especially canthaxanthin and zeaxanthin, was reduced in the fusion enzyme strains (Figure 6B and Figure 9). This suggested that the fusion enzyme accelerated the conversion of canthaxanthin and zeaxanthin to astaxanthin. Unexpectedly, the intermediate 3'-hydroxyechinenone significantly accumulated in the fusion enzyme strain (Figure 6B and Figure 9).Although the fusion enzyme was more efficient in producing astaxanthin compared to the individual enzymes, the accumulation of intermediates was not effectively addressed. Therefore, a key challenge was to explore how to maximize the flux from β-carotene to astaxanthin synthesis without the accumulation of intermediates.

[0110] Targeting enzyme fusions to intracellular compartments enhances astaxanthin biosynthesis The chromatographic profile of carotenoids synthesized by strains harboring the fusion enzyme CrtW-Z (Figure 6B) showed that precursor β-carotene and ketocarotenoid intermediates (echinenone, 3'-hydroxyechinenone, and zeaxanthin) accumulated, impeding carbon flux to astaxanthin synthesis. To reduce the formation of these intermediates, we investigated the effect of increasing the copy number of the astaxanthin biosynthetic pathway by introducing additional copies of the fusion enzyme CrtW-Z into the astaxanthin-producing strain YL11 to generate strain YL12. However, this did not affect the amount of astaxanthin produced (Figure 10A), and the accumulation of intermediates remained largely unaddressed (Figure 10B). This led us to hypothesize that the inefficient production of astaxanthin is likely due to the precursor β-carotene being sequestered within lipid droplets and not readily accessible to the cytosolic enzymes that convert it to astaxanthin.

[0111] The lipid body (LB) of Y. lipolytica actually forms a hydrophobic pocket that can preferentially sequester lipophilic isoprenoid compounds. As the astaxanthin precursor β-carotene is such a lipophilic molecule, we investigated whether its sequestration in LBs (Figure 11) would affect astaxanthin production. We hypothesized that targeting the astaxanthin pathway to LBs would accelerate the conversion of β-carotene to astaxanthin due to increased physical proximity between the enzyme and the substrate. To this end, we took advantage of a well-characterized N-terminal localization signal that can target proteins to LBs. The fusion enzyme CrtW-Z was targeted to LBs by linking it with the oleosin sequence of the protein position tag (Figure 7A), providing an alternative biological pathway for astaxanthin biosynthesis. As expected, the LB-targeted strain YL13 showed a titer of 46.8 mg / L of astaxanthin, a 1.62-fold increase compared to the control strain expressing the pathway in the cytosol (Figure 7D). Most notably, the accumulation of β-carotene and ketocarotenoid intermediates was reduced (Figures 7B-7C).

[0112] Inspired by these results, the intracellular sites of β-carotene synthesis were exploited. Prediction of the transmembrane helices of heterologous proteins involved in β-carotene biosynthesis (GGPPsa, CarB, and CarRP) revealed that the other two enzymes, except GGPPsa, possess transmembrane helices (Figure 12A-12C), suggesting that CarB and CarRP are localized in intracellular organelles. To investigate the intracellular localization sites of these three proteins, a fluorescence assay was employed from the colocalization of fusion proteins with GFP, and it was found that both CarB and CarRP were localized in the endoplasmic reticulum (ER), while GGPPsa was localized in the cytosol (Figure 13). This indicated that β-carotene biosynthesis mainly occurred in the ER, consistent with the synthesis of triacylglycerides (TAGs) that are synthesized in the ER and further aggregated in LBs (Figure 7A) (Xu et al., 2016). Thus, the ER provides another potential target for compartmentalization of the astaxanthin pathway.

[0113] Besides LBs and ER, another organelle that may affect β-carotene accumulation in cells is the peroxisome, which also serves as a storage compartment for lipophilic compounds (Liu et al., 2020). Thus, the astaxanthin pathway was targeted to the ER, as expressed by the fusion enzyme CrtW-Z, and the peroxisome was targeted by the fusion of the well-characterized targeting sequences KDEL and SKL, respectively (Figure 7A). As expected, the corresponding organelle-targeted strains significantly increased astaxanthin titers compared to the cytosolic pathway-expressing strains (Figures 7B-7D). The YL14 strain harboring the engineered ER-targeted pathway produced 53.2 mg / L astaxanthin after 72 h of culture, a 1.84-fold increase compared to its cytosolic counterpart (Figure 7D). The corresponding peroxisome-targeted strain YL15 produced approximately 58.7 mg / L astaxanthin, a 2.03-fold increase compared to strain YL11 with cytosolic pathway expression (Figure 7D).

[0114] Considering that a significant portion of the β-carotene pool resides in the ER, LB, and peroxisomes, we considered targeting the pathway to multiple compartments. We found that simultaneous targeting of the fusion enzyme CrtW-Z to LB and ER significantly increased astaxanthin titers compared to those of a single subcellular compartment (Figure 7D). Notably, the bottleneck manifested by intermediate accumulation of β-carotene and ketocarotenoids was effectively alleviated (Figures 7B-7D). Furthermore, in the triple-organelle engineered strain YL17 with an additional CrtW-Z localized to peroxisomes, astaxanthin titers were further increased up to 139.4 mg / L (Figure 7D). Taken together, these results support that compartmentalization not only allows intermediates to be accessible to downstream engineered biocatalysts, but also enhances the catalytic activity of enzymes through a unique physicochemical environment.

[0115] Optimization of fermentation conditions to maximize the conversion of β-carotene to astaxanthin Next, we investigated the effect of medium composition on astaxanthin production. The glucose concentration was varied while keeping the nitrogen amount constant, effectively changing the C / N ratio of the medium. Astaxanthin production was observed to vary significantly with the initial glucose concentration. Astaxanthin titer gradually increased with increasing glucose concentration, reaching the highest level in YPD40 medium (Figure 8A). However, the yield of astaxanthin decreased with increasing initial glucose concentration (Figure 8A). Furthermore, a clear positive correlation was observed between the initial glucose content and the accumulation of intermediates, including β-carotene and ketocarotenoid intermediates (Figure 14). This is probably due to the increase in lipid content caused by the increase in C / N ratio, which resulted in an increase in β-carotene sequestered in the fat body, limiting LB target enzymes and resulting in intermediate accumulation. Based on these results, YPD20 medium was selected for the demonstration of astaxanthin production in fed-batch fermentation. The YL17 strain achieved a total astaxanthin titer and content of 858 mg / L and 16.7 mg / gDCW, respectively, in flask fed-batch fermentation (Figure 8B), which are the highest values ​​reported so far in yeast. Furthermore, the performance of the constructed strain was evaluated by scaling up the fermentation to a 3-L bioreactor. However, only a titer of 453 mg / L of astaxanthin was obtained (Figure 8C), suggesting that further optimization of fermentation in fermenters will be required in the future.

[0116] Simultaneous immobilization of the enzyme in all three organelles resulted in the greatest increase in astaxanthin synthesis, ultimately producing 858 mg / L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the starting strain). It is expected that the methods and products disclosed herein will maximize the potential of intracellular compartmentalization and help advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.

[0117] As mentioned above, the present disclosure relates to the fusion expression of two key enzymes in the astaxanthin pathway and the performance of the fusion when targeting various subcellular compartments. The activities of the key enzymes β-carotene ketolase (CrtW) and hydroxylase (CrtZ) from different sources were evaluated, and the pair PsCrtW / HpCrtZ (originating from Paracoccus sp. and Haematococcus pluvialis, respectively) was optimal for astaxanthin accumulation. The activities of PsCrtW and HpCrtZ were combined by the creation of an enzyme fusion to overcome the leakage of non-endogenous intermediates. Finally, the above astaxanthin biosynthetic pathway of the fusion enzymes was targeted to the intracellular compartment of the fat body (LB), alone as well as in combination with compartmentalization in the endoplasmic reticulum (ER) and peroxisomes. Compared to the cytosolic pathway, targeting the astaxanthin pathway to organelles resulted in a significant increase in production and a decrease in intermediate accumulation. Furthermore, targeting the astaxanthin pathway to all three of the LBs, ER, and peroxisomes simultaneously resulted in the highest production of astaxanthin, ultimately achieving 858 mg / L (16.7 mg / g DCW) in fed-batch fermentation. These results demonstrate the potential of Y. lipolytica for lipophilic metabolite production by targeting pathway expression to subcellular compartments that enable efficient functioning of the biosynthetic pathway.

[0118] We deployed these strategies described herein to maximize the production of the carotenoid astaxanthin in Y. lipolytica. First, we generated functional fusions of β-carotene ketolase and hydroxylase (CrtW-Z or CrtZ-W) and showed that astaxanthin production was increased above the levels achieved by the individually expressed enzymes (CrtW+Z). The largest differences in intermediate accumulation between the individually expressed and fused enzymes were in the profiles of canthaxanthin and zeaxanthin in the CrtW+Z strain and 3'-hydroxyechinenone in the CrtW-Z strain. Canthaxanthin or zeaxanthin are synthesized from β-carotene in two enzymatic steps that require only CrtW or CrtZ, respectively, whereas the production of 3'-hydroxyechinenone requires the participation of both enzymes. This quantitative change in the composition of intermediates between the CrtW+Z and CrtW-Z strains may indicate that canthaxanthin and zeaxanthin are more readily converted to downstream metabolites upon fusion of both. The reduced intermediate leakage and accelerated overall reaction rates highlight the enhanced enzyme interactions upon fusion.

[0119] Example 4. Materials and methods related to Example 3 Culture conditions and media E. coli DH5α cells were grown in Luria-Bertani (LB) medium (BD bioscience) at 37°C with constant shaking. The corresponding antibiotics (100 μg / mL ampicillin and 50 μg / mL kanamycin) were added for plasmid selection. All Y. lipolytica strains were cultured at 30°C with shaking at 230 rpm. For Y. lipolytica, YPD medium consisting of 10 g / L yeast extract (BD bioscience), 20 g / L peptone (BD bioscience), and 20 g / L glucose (Sigma-Aldrich) was used. In addition, YNB medium, consisting of 1.7 g / L yeast nitrogen base (YNB, VWR Life Science), 20 g / L glucose, 5 g / L ammonium sulfate, 15 g / L agar (BD bioscience), and 0.77 g / L of the appropriate complete supplement mixture without uracil, leucine, or tryptophan (Sunrise science products), was used for selection of transformed Y. lipolytica strains.

[0120] Plasmid and strain construction E. coli DH5α was used for cloning and plasmid propagation. The po1f strain of Y. lipolytica was used as the base strain, and all derivatives and plasmids constructed are listed in Table 2. Primers used for plasmid construction are shown in Table 3. All restriction enzymes were purchased from New England Biolabs (NEB). PCR amplification was performed using Q5 high-fidelity DNA polymerase (NEB) or GoTaq DNA polymerase (Promega). PCR fragments were purified using ZYMO Fragment Recovery Kit (ZYMO research). Plasmids were then constructed from the purified PCR fragments using Gibson Assembly kit (NEB) and transformed into chemically competent E. coli cells by heat shock and extracted using QIAprep Spin Miniprep Kit (Qiagen). All procedures were performed according to the manufacturer's instructions. All engineered Y. lipolytica strains were constructed by transforming linearized plasmids (Not1 digested) using the lithium acetate method. Recombinants were verified by PCR amplification from genomic DNA. All astaxanthin biosynthetic genes evaluated in this study were codon-optimized for Y. lipolytica.

[0121] Curation of auxotrophic markers using the Cre-loxP system Plasmid pYLMA-Cre was transformed into the target Y. lipolytica strain to rescue the URA3, LEU2, and TRP1 markers. Transformants were selected on YPD agar plates supplemented with hygromycin B at a final concentration of 250 mg / L (Sigma-Aldrich). After 2–3 days of culture, colonies were transferred to new YPD plates containing hygromycin B for another day to ensure further successful marker removal. Marker curation was confirmed by subculturing colonies on YNB-Ura, YNB-Leu, and YNB-Trp agar plates, respectively. Successful removal of all three markers results in a phenotype exhibiting uracil, leucine, and tryptophan deficiency. The positive strains were then cured of plasmid pYLMA-Cre in the cells by incubating them on YPD agar plates at 30 °C for 24 h in two to three replicates.

[0122] Shake flask fermentation A single colony of the recombinant strain was picked from the plate, inoculated into 2 mL of YPD medium, and grown overnight (16–18 h) at 30 °C and 230 rpm. The culture was then transferred to 10 mL of YPD medium (initial OD 600 = 0.1) and cultured at 30°C and 230 rpm for 3 days.

[0123] Quantification of residual glucose in the medium For quantification of residual glucose, 500 μL samples were extracted from the cultures. Cells were centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a 0.2 μm syringe filter before being injected into an Agilent technologies 1260 high performance liquid chromatography (HPLC) equipped with a refractive index detector. A Bio-Rad HPX-87H column was used for separation, with 14 mM sulfuric acid as the mobile phase at a rate of 0.7 mL / min. The injection volume was 10 μL. The column temperature was 50 °C.

[0124] Carotenoid Extraction Carotenoid extraction was performed as described with the following modifications. Briefly, 50–100 μL of culture was centrifuged at 12,000 g for 1 min and the cell pellet was suspended in 900 μL of dimethyl sulfoxide (DMSO) and then heated at 50 °C for 1 h in a water bath until the cells were bleached. The DMSO extract was briefly mixed with 450 μL of methanol and centrifuged at 14,000 g for 5 min. The resulting supernatant was transferred to glass vials for analysis and quantification of carotenoids.

[0125] Carotenoid Analysis and Quantification Carotenoid production was expressed as grams per liter of fermentation broth (g / L) and milligrams per gram of dry cell weight (mg / g DCW). Optical density was measured at 600 nm using a Thermo Spectronic Genesys 20 (Thermo Scientific) and used to calculate cell mass (DCW = 0.30 × OD 600 , Figure 15). Analysis and quantification of astaxanthin was performed by HPLC (SHIMADZU LC-20 AT) equipped with a Kromasil C18 column (4.6 mm x 250 mm) and UV / VIS detection at 475 nm. The mobile phase consisted of acetonitrile-methanol-isopropanol (5:3:2 v / v) with a flow rate of 1 mL / min at 40 °C. A standard curve of astaxanthin (Sigma-Aldrich) was generated by running the same extraction process as the samples.

[0126] Targeting biosynthetic pathways to subcellular compartments The fusion enzyme CrtW-Z-dependent astaxanthin biosynthetic pathway was targeted to different subcellular compartments (ER, LB, and peroxisomes) using specific addressing signals. Enzymes involved in the astaxanthin biosynthetic pathway were directed to the ER by adding a C-terminal KDEL utilizing the following nucleotide sequence 5'-AAGGACGAGCTG-3' (SEQ ID NO: 6) while removing the termination codon at the end of the targeting signal. Similarly, peroxisome or LB targeting of proteins was ensured by the addition of SKL (nucleotide sequence 5'-TCCAAGCTG-3') or oleosin from Zea mays (codon-optimized oleosin sequence is listed in Table 4), as previously performed in other studies on the engineering of Y. lipolytica (Yang et al., 2019). The fusion enzyme CrtW-Z without a targeting signal was directed to the cytoplasm.

[0127] Fed-batch fermentation Bioreactor fed-batch fermentations were performed in 3 L fermenters (New Brunswick Bioflo115 system). The first fermentation was performed in 1 L medium containing 100 g / L glucose, 100 g / L peptone, and 50 g / L yeast extract. The temperature was maintained at 30 °C. Dissolved oxygen was controlled at 20% saturation with an agitation cascade at 250-800 rpm. Air was injected into the fermenter at 2 vvm. pH was maintained at 6.8 by feeding 5 M NaOH or 5 M HCL. Foaming was prevented by the addition of antifoam 204 (Sigma-Aldrich). The fed-batch operation was started after 72 h of cultivation in 15 x YPD medium. Samples were taken every 24 h to measure OD 600 , glucose and astaxanthin concentrations were measured. Flask fed-batch fermentations were carried out in 50 mL conical flasks with a working volume of 10 mL YPD medium. 15 x YPD medium was fed every 48 hours and pH was not controlled.

[0128] [Table 1] [Table 2]

[0129] [Table 3-1] [Table 3-2] [Table 3-3]

[0130] [Table 4] [Table 5]

[0131] Example 5. Removal of lycopene substrate inhibition allows high carotenoid productivity in Yarrowia lipolytica Substrate inhibition of enzymes can be a major obstacle for the production of valuable chemicals in engineered microorganisms. Substrate inhibition of lycopene cyclase was identified as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, two independent approaches were used. Structure-guided protein engineering generated the variant Y27R, characterized by a complete loss of substrate inhibition without reducing the enzyme activity. Alternatively, the establishment of a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevented the onset of substrate inhibition by redirecting metabolic flux away from the inhibiting metabolite while maintaining sufficient flux towards product formation. Both approaches were highly effective, resulting in high levels of almost exclusive β-carotene production. Finally, a strain capable of producing 39.5 g / L of β-carotene (a 1441-fold improvement over the initial strain) in bioreactor fermentation was constructed, with a productivity of 0.165 g / L / h. The findings described herein provide an effective approach for removing substrate inhibition in engineering pathways for the efficient synthesis of natural products.

[0132] Engineering microbes to produce valuable chemical products is an attractive alternative to sourcing these compounds from nature or extracting them from petrochemicals via chemical synthesis (Chen and Nielsen., 2013; Li and Smolke, 2016; Liu and Nielsen, 2019). However, synthetic biology efforts to achieve economically viable and scalable titers and productivity are frequently hindered by undesirable regulatory mechanisms that modulate the activity of enzymes. Such mechanisms have evolved to mediate optimal cellular responses to changing physiological conditions, but they also pose significant obstacles in redirecting metabolic flux from native growth-optimizing pathways to engineered, desirable metabolic pathways. This problem is particularly pronounced for compounds that require long and complex synthetic pathways (e.g., isoprenoids), which frequently create bottlenecks that can reduce cellular fitness and pathway productivity (Wu et al., 2016; Keasling et al., 2010). Therefore, it is essential to develop methods to avoid the effects of enzyme inhibition in constructing highly productive and robust strains.

[0133] Substrate inhibition is one such regulation of enzyme levels that is deployed in cells to optimize cellular economy and maximize growth in response to temporal variations in the environment (Reed et al., 2010). Moreover, such mechanisms are often used to design treatments for various diseases (Boutin et al., 2005; Belzer et al., 2013). However, this is undesirable in industrial applications of microorganisms for product synthesis mediated by enzymatic reactions. Enzyme inhibition is typically triggered when the substrate concentration exceeds a certain threshold, thus preventing catalytic conversion of the substrate and limiting flux through the desired pathway. Substrate inhibition is therefore particularly detrimental to the synthesis of the desired end product when present in the middle of a metabolic pathway, causing accumulation of intermediates, disruption of the pathway, and changes in the profile of the products formed. Several methods have been explored to address this limitation, such as enzyme immobilization (Singh et al., 2013; Matwo et al., 2004), two-phase partitioning bioreactor systems (Daugulis et al., 2011; Gao et al., 2009; Nielsen et al., 2009), batch substrate feeding strategies (Kim et al., 2007), and protein engineering (Chen et al., 2014; Shang et al., 2020), however, most of these solutions are limited to systems where inhibition is provided by the starting substrate and are difficult to apply in the context of microbial engineering for chemical production.

[0134] Using the oleaginous yeast Yarrowia lipolytica for the overproduction of carotenoids, two independent strategies have been demonstrated that almost completely circumvent substrate inhibition. First, the enzyme lycopene cyclase was identified as a bottleneck in carotenoid synthesis due to strong substrate inhibition by lycopene. This resulted in low titers of β-carotene as well as high amounts of lycopene as a by-product. In light of this, the first strategy was to generate protein variants with reduced inhibition using structure-guided protein design combined with phylogenetic information. Among the 50 variants generated, a single mutation, Y27R, was identified that completely abolished substrate inhibition without reducing enzyme activity, resulting in a marked increase in β-carotene production and a selectivity of 98% (% of product over the sum of all carotenoids). Alternatively, the second approach yielded similar titers and selectivity for β-carotene by reducing the carbon flow through the carotenoid pathway, thus preventing the accumulation of inhibitory metabolites to inhibitory levels, contrary to the traditional paradigm of pathway engineering. This was achieved by establishing a metabolic flow restrictor through geranylgeranyl pyrophosphate synthase (GGPPS) that regulates the rate of formation of the substrate lycopene, thereby effectively alleviating substrate inhibition. Although this approach reduces the flux through the pathway of interest, the benefits of maintaining high enzyme activity at suppressed substrate levels more than compensated for the productivity loss due to flux diversion. Using the above method and by carefully partitioning cell resources dedicated to carotenoid synthesis versus conservation, a strain was finally established that could produce 39.5 g / L of β-carotene (98% selectivity) with a volumetric productivity of 0.165 g / L / h in bioreactor fermentation. Moreover, deliberate exploitation of the substrate inhibition effect shifted the product profile toward lycopene, achieving a lycopene titer of 17.6 g / L and a productivity of 0.073 g / L / h.

[0135] Substrate inhibition of lycopene cyclase limits carotenoid synthesis. The synthesis of β-carotene in Y. lipolytica requires the heterologous expression of three genes encoding the enzymes phytoene synthase, phytoene dehydrogenase, and lycopene cyclase (Figures 16A and 22). In addition, geranylgeranyl diphosphate synthase (GGPPS) should also be considered, since it controls the flux towards carotenoid synthesis rather than sterols (Figures 16A and 22). The relevant genes were obtained for expression from the eukaryotes Xanthophyllomyces dendrorhous and Mucor circinelloides. Since Y. lipolytica already harbors a natural copy of GGPPS, the first step was to introduce gene expression cassettes encoding phytoene dehydrogenase and a bifunctional phytoene synthase / lycopene cyclase from X. dendrorhous (CrtI and CrtYB, respectively) (Verdoes et al., 1999; Verdoes, Krubasik et al., 1999) or M. circinelloides (CarB and CarRP, respectively) (Velayoes, Blasco et al., 2000; Velayos, Eslava et al., 2000) into the TRP1-disrupted Y. lipolytica po1f strain (po1f-T) (Figure 23 and Table 6). Strain YLMA02 expressing the enzyme from M. circinelloides produced 4.12-fold more β-carotene (27.4 mg / L) than strain YLMA01 expressing the enzyme from X. dendrorhous (Figure 16B). Therefore, the CarB / CarRP pair was used in all subsequent studies.

[0136] Although the synthesis of β-carotene was observed in YLMA02, the titer was very low, leading us to consider the GGPPS step as the next target. Introducing an additional copy of GGPPS from X. dendrorhous (GGPPxd) into strain YLMA02 significantly increased the titer of β-carotene to 0.48 g / L (Figure S16C). However, this was accompanied by a large increase in lycopene accumulation (Figure S16C), suggesting that the cyclization of lycopene to β-carotene is the bottleneck of the pathway. Accordingly, the expression of lycopene cyclase was increased by increasing its gene copy number. Moreover, since the cyclase activity is conferred by the R domain of the bifunctional enzyme CarRP (Velayos, Eslava et al., 2000), a modified version of the protein with the P domain deleted or mutated was also introduced (Figure S24A) to function as a dedicated cyclase. None of these efforts succeeded in improving β-carotene titers (Figure 24B), even though higher mRNA levels were observed (Figure 24C). We next examined the effect of introducing lycopene cyclases from four other organisms (EuCrtY, PaCrtY, PfCrtY, and HpCrtY) (Table 6); this slightly improved β-carotene production (Figure 24D). However, the issue of lycopene accumulation was hardly addressed, suggesting that overexpression of lycopene cyclase was not an effective strategy, regardless of its origin. The protein level of lycopene cyclase was very likely not a limitation of this study.

[0137] Since lycopene was the only aggregate precursor (Figure 25) and adding various copies of lycopene cyclase did not circumvent the problem, it was hypothesized that the activity of lycopene cyclase was inhibited by excess lycopene due to substrate inhibition (Figure 16A). To test this hypothesis, a possible correlation between lycopene cyclase activity and lycopene concentration was examined using an in vitro enzyme assay with a yeast microsome system, since CarRP is predicted to be a membrane protein with six transmembrane helices (Figure 26). The results showed that lycopene cyclase activity was biphasic with respect to lycopene concentration: the enzyme activity initially increased with lycopene concentration and then decreased when lycopene reached high concentrations (Figure 16D). This supported the hypothesis that lycopene cyclase inhibited the substrate, creating a major bottleneck in carotenoid biosynthesis.

[0138] Structure-guided protein engineering completely removes substrate inhibition Next, we evaluated the removal of the substrate inhibition effect of lycopene cyclase by protein engineering. As no crystal structure was available, we used the Transform-restrained Rosetta (TrRosetta) platform (Yang et al., 2020) to create a computational model of the R domain of CarRP (lycopene cyclase) (Figure 27). We used evolutionary information to generate Position Specific Scoring Matrices (PSSMs) from multiple sequence alignments to identify mutable positions to deconvolute regions of the enzyme that affect substrate inhibition. Single and double amino acid substitutions were made based on PSSM information and clustered to ensure maximum spread of the tested variants (Figure 28). The variants were calculated between-sequence distances using PAM30 and sequences were subdivided using agglomerative clustering to maximize the information obtained during the initial screening. This method generated a set of 50 candidates with mutations spread throughout the enzyme (Figure 17A). For each variant, selectivity for β-carotene was obtained (Figure 17B). Among them, three variants, Y27R, V175W, and T31R-F92W, showed a significant increase in β-carotene selectivity and improved production metrics (Figures 17B-17C) without affecting gene expression (Figure 29), suggesting alleviation of substrate inhibition effects. All substitutions in the three mutants were located in specific parts of the enzyme (Figure 30), with the loss of inhibition being the most pronounced for Y27R. Variant Y27R demonstrated a complete loss of substrate inhibition without a decrease in enzyme activity (Figure 16D), resulting in a titer of 2.38 g / L β-carotene (Figure 17C) and 98% selectivity (compared to 18% of the wild type, Figure 17D).

[0139] We next investigated whether the β-carotene pathway containing variant Y27R could maintain the property of minimizing substrate inhibition in the presence of significantly higher precursor / substrate formation rates. To this end, four key enzymes of the mevalonate (MVA) pathway (Ro et al., 2006; Westfall et al., 2012) (Figure 22), tHMGR, ERG12, IDI, and ERG20, were overexpressed in the β-carotene-producing strain YLMA11 expressing Y27R (Figure 17E). As a result, the titer of β-carotene was increased to 3.43 g / L while maintaining a high selectivity of 97.8%. Furthermore, a synthetic isopentenol utilization pathway (IUP) (Chatzivasileiou et al., 2019; Clomburg et al., 2019; Rico et al., 2019; Lund et al., 2019) was introduced via expression of choline kinase (CK) and isopentenyl phosphate kinase (IPK) (Figure 22), which further increased β-carotene production (4.22 g / L) by 23% without compromising selectivity (YLMA15, Figure 17E). These results demonstrate that the reconstituted non-substrate inhibitory pathway functions efficiently in high isoprenoid flux strains without being affected by intracellular levels of intracellular precursors / substrates.

[0140] Management of substrate inhibition of lycopene cyclase by a GGPPS-mediated flux flow restrictor Other viable options were also explored that could eliminate substrate inhibition without the need to modify lycopene cyclase. It was hypothesized that attenuating the rate of lycopene formation relative to its conversion rate could lower the intracellular concentration of lycopene below inhibitory levels. However, this would need to be well tuned to prevent excessive attenuation of lycopene formation, which would result in an overall decrease in the production rate of the end product. To this end, a metabolic flow restrictor was created using the branching point of the FPP node to regulate the flux to lycopene to remain below inhibitory levels while maintaining a high conversion of lycopene to β-carotene (Figure 18A). Therefore, we searched for GGPPS mutants with various activities by screening five different enzymes with different catalytic efficiencies measured by the in vivo GGPP synthesis rate (Table 6) (Figure 18B). Compared to GGPPxd, the other four GGPPSs were less productive (Figure 18B), which should lead to a decrease in the lycopene synthesis flux. Introducing these low activity GGPPS into strain YLMA02 (basal strain harboring wild-type CarRP) reduced lycopene levels (Figure 18C) and increased β-carotene production, reaching up to 1.26 g / L with 92.5% selectivity when using GGPPsa from Sulfolobus acidocaldarius (Figure 18C). This was further confirmed by the time course of lycopene and β-carotene concentrations (Figure 18D). Interestingly, expression of the attenuated GGPPsa (YLMA25), although it slightly reduced the flux involved in carotenoid synthesis, avoided substrate inhibition and allowed an overall balanced pathway directing all carbon flux towards β-carotene formation, allowing this pathway to behave similarly to the Y27R mutant (YLMA11, Figure 18D). In contrast, the highly efficient GGPPxd-expressing strain YLMA03 led to excessively rapid accumulation of lycopene, causing substrate inhibition and preventing its conversion to β-carotene (Figure S18D). These results indicate that the GGPPS-mediated metabolic flow restrictor effectively alleviates substrate inhibition and regulates the flux of lycopene through both the upstream and downstream pathways for optimal conversion to β-carotene.

[0141] To bridge the gap in β-carotene production between the two engineering strategies to alleviate substrate inhibition (Figure 17C and Figure 18C), the GGPPS-mediated flux flow restrictor-supported pathway was overexpressed by the insertion of an additional copy (Figure 18E). This regulation not only enhanced β-carotene biosynthesis up to 2.13 g / L, but also further maximized its selectivity (from 92.1% to 98.1%, Figure 18E). Moreover, similar to previous results, overexpression of MVA and IUP promoted β-carotene production up to 3.72 g / L without affecting selectivity (Figure 18F). This further highlighted the fact that this pathway can function with high efficiency once the substrate inhibition issue is resolved.

[0142] The product profile of β-carotene versus lycopene can be shifted by altering GGPPS activity in vivo (Figure 18B-18C). The high activity of GGPPxd can also be exploited to reconstitute a dedicated lycopene producing strain. Combining this idea with the CarRP variant (E78K) (Velayos, Eslava, et al., 2000) (Figure 32A-32B), we successfully produced 2.62 g / L of lycopene with undetectable amounts of β-carotene (Figure 18G). Further increase in lycopene production was achieved by overexpressing the MVA pathway and introducing the IUP, reaching a titer of 3.09 g / L (YLMA34, Figure 33).

[0143] Partitioning carbon flux between isoprenoid and lipid synthesis to enhance intracellular carotenoid accumulation The lipid body of Y. lipolytica creates a hydrophobic pocket that facilitates the sequestration and storage of lipophilic isoprenoid products (Qiao et al., 2017). However, increasing the supply of triacylglycerol (TAG) enhances isoprenoid storage (Ma et al., 2019; Larroude et al., 2018), at the expense of acetyl-CoA, a common precursor for isoprenoid and lipid synthesis (Figure 19A). Thus, carbon flux needs to be optimally partitioned between lipid and isoprenoid synthesis, with the goal being to ensure a sufficient supply of lipids to encapsulate the produced isoprenoids while not drawing too much acetyl-CoA away from the MVA pathway. For this purpose, we decided to cultivate the β-carotene producing strain YLMA15 in media with various carbon-nitrogen (C / N) ratios (Figure 34); this is because the lipid formation capacity can be easily controlled by the C / N ratio of the culture medium (Braunwald et al., 2013; Somashekar et al., 20000). The initial glucose concentration for all conditions was fixed at 50 g / L, which was determined to be optimal for the strain (Figure 35). With increasing C / N ratio, the cellular lipid content was found to increase monotonically (Figure 19B), while the total biomass decreased (due to the reduced nitrogen availability; Figure 36). However, the optimal conditions for β-carotene production were found to be in YLMA15 with a C / N ratio of 9:1, both in terms of titer (7.5 g / L, Figure 19B) and cell content (360.8 mg / g DCW, Figure 19D). 10 P 10 D 50 The optimum Y-carotene concentration was obtained by using a medium containing 10 g / L yeast extract, 10 g / L peptone, and 50 g / L glucose. Deviations from this optimum resulted in a decrease in β-carotene concentration, which is consistent with the hypothesis and highlights the importance of an optimal balance between carotenoid and lipid biosynthesis. 10 P 10 D 50 The medium was also applied to a lycopene producing strain (YLMA34) and a concentration of 8.02 g / L of lycopene was obtained after 5 days of fermentation (Figure 37).

[0144] Carotenoid biosynthesis during glucose-depleted stationary phase is supported by cellular lipid degradation In the fermentation experiments with the YLMA15 strain described herein, we found that the content of β-carotene continued to rise during stationary phase even after glucose in the medium was exhausted (Figure 20A). This is likely because Y. lipolytica mobilized previously stored TAG as an alternative carbon source to support carotenoid formation (Wang et al., 2020). To test this hypothesis, we decided to characterize the cells and their intracellular lipids both before and after glucose depletion. Throughout the fermentation process, the lipid content initially increased, reaching a maximum on day 3, and then rapidly decreased once glucose was completely consumed (Figure 20A). However, despite the decrease in lipid content, the β-carotene content continued to rise well beyond the point of glucose exhaustion (Figure 20A), suggesting that TAG is utilized to maintain metabolic activity and carotenoid synthesis. Microscopic visualization of the changes that occurred during fermentation was consistent with this hypothesis. During the first 3 days, when glucose was still present, the intracellular lipid droplets gradually aggregated into lipid bodies, sequestering the produced β-carotene (Figure 38). However, the lipid bodies disappeared in the later stages of fermentation due to the degradation of TAG, and the accumulated β-carotene was then dispersed throughout the cells (Figure 38).

[0145] Degradation of TAG occurs mainly via β-oxidation to generate acetyl-CoA (Xu et al., 2016) (Figure 19A), so it is likely to provide the carbon skeleton for β-carotene synthesis during glucose-starved stationary phase. To test this hypothesis, YLMA15 was uniformly labeled with [U- 13C6] were cultured in YNB medium with glucose and natural stearic acid. Upon addition of stearic acid, which is catabolized by β-oxidation, a large proportion of unlabeled IPP / DMAPP and GGPP, the main precursors of β-carotene synthesis, was observed within 24 hours of culture (Figure 20B-20C). These results suggest that acetyl-CoA formed by β-oxidation may indeed support the MVA pathway and ultimately contribute to β-carotene formation. Therefore, this is probably the mechanism that cells use to convert TAG to carotenoids during glucose-depleted stationary phase.

[0146] Bioreactor culture research Finally, the performance of the constructed strains was evaluated, where substrate inhibition was alleviated in 3L fed-batch culture. After bioreactor optimization, strain YLMA15 yielded total β-carotene titer and content of 39.5 g / L and 494 mg / g DCW, respectively, with a productivity of 0.165 g / L / h (Figures 21A-21C). Similarly, bioreactor fermentation of lycopene-producing strain YLMA34 yielded 17.6 g / L (313 mg / g DCW) of lycopene with a productivity of 0.073 g / L / h (Figures 21D-21F). Notably, no change was observed in the selectivity for β-carotene of strain YLMA15 during the scale-up process (98% in fed-batch bioreactor and 97.9% in shake flasks (Figure 39)). These figures demonstrate the robustness of the engineering strategy in large culture volumes and high cell density fermentations, with production indices exceeding previous results (a summary of reported carotenoid production is shown in Table 8).

[0147] This disclosure concerns the demonstration that lycopene cyclase inhibits β-carotene production by substrate inhibition, although this regulatory effect has not been widely reported in the context of microbial synthesis. Substrate inhibition of enzymes can be overcome by modification of the protein structure, a strategy that has been successfully applied to many enzymes (Shang et al., 2020). However, these efforts rely on readily available protein crystal structures, which is not the case for lycopene cyclase investigated here. Directed evolution is a powerful method to adapt enzymes to specific tasks (Reetz et al., 2013), but screening large libraries often requires high-throughput detection methods. Furthermore, due to the lack of crystal structure information, efficient structure-driven design is highly dependent on both the quality of the computational model and the accuracy of docking the substrate into its binding site. Here, we refined the search by combining structural and phylogenetic information and were able to isolate promising mutants by screening only 50 variants, three of which showed reduced or removed substrate inhibition. Furthermore, information from key amino acids can be iteratively fed back into the computational model to further optimize enzyme properties. A mechanistic understanding of what triggered the removal of substrate inhibition requires more thorough investigation beyond the scope of this study. The small number of protein variants designed and tested suggests that a structure-guided approach combined with phylogenetic information may provide an effective strategy for protein engineering.

[0148] The degree of substrate inhibition can also be controlled by tuning the relative rates of upstream and downstream pathways that form and consume the inhibiting substrate. In the case of β-carotene synthesis, selection of a less active GGPPS variant reduced flux through the carotenoid pathway. However, the resulting abolition of substrate inhibition allowed all carotenoid flux to be diverted to β-carotene synthesis rather than to a combination of both lycopene and β-carotene. This increased β-carotene production with high specificity (>98%) despite low GGPPS activity. On the other hand, substrate inhibition can also be purposefully exploited when lycopene is the product of interest. In this case, a highly efficient GGPPS would result in lycopene formation exceeding its depletion, leading to lycopene accumulation, with the imbalance further amplified by substrate inhibition. Correspondingly, the product profile shifts dramatically from β-carotene-rich to lycopene-rich. These findings indicate that manipulation of proximal enzymes can profoundly affect pathway dynamics and provide a new paradigm for controlling metabolism.

[0149] Another important consideration in metabolic engineering is how the heterologous pathway interacts with the native pathway. Designing pathways that are orthogonal or have minimal impact on the native function of the organism has been the focus of many strain engineering efforts (Ro et al., 2006; Tan et al., 2016; Zhao et al., 2018; Brockman et al., 2015). It is well known that the lipophilicity of carotenoids promotes their storage in the lipid body of the cell. Larroude et al. (Larroude et al., 2018) found that an engineered lipid overproducer strain produced more β-carotene at a titer of 6.5 g / L, accompanied by the simultaneous production of 42.6 g / L of lipids. While providing a compatible compartment for hydrophobic isoprenoid accumulation, the de novo formation of TAG also consumes large amounts of carbon sources, thus limiting acetyl-CoA flux to MVA and product formation pathways. Here, we demonstrated that the intrinsic TAG accumulation capacity of Y. lipolytica was sufficient for carotenoid sequestration, and by balancing the flux distribution between carotenoid and lipid synthesis through tuning the C / N ratio, a large proportion of the acetyl-CoA pool was reserved for carotenoid production, leading to higher titers and contents per cell.

[0150] Example 6. Materials and methods related to Example 5 Culture conditions and media E. coli DH5α cells were grown in Luria-Bertani (LB) medium (BD bioscience) at 37°C with constant shaking. The corresponding antibiotics (100 μg / mL ampicillin and 50 μg / mL kanamycin) were added for plasmid selection. All Yarrowia lipolytica strains were cultured at 30°C with shaking at 230 rpm. For Y. lipolytica, YPD medium consisting of 10 g / L yeast extract (BD bioscience), 20 g / L peptone (BD bioscience), and 20 g / L glucose (Sigma-Aldrich) was used. In addition, YNB medium, consisting of 1.7 g / L yeast nitrogen base (YNB, VWR Life Science), 20 g / L glucose, 5 g / L ammonium sulfate (VWR Life Science), 15 g / L agar (BD bioscience), and 0.77 g / L of the appropriate complete supplement mixture without uracil, leucine, or tryptophan (Sunrise science product), was used for selection of transformed Y. lipolytica strains.

[0151] Plasmid and strain construction E. coli DH5α was used for cloning and plasmid propagation. The po1f strain of Y. lipolytica was used as the base strain, and all derivatives and plasmids constructed in this study are listed in Table 7. The primers used for plasmid construction are shown in Table 9. All restriction enzymes were purchased from New England Biolabs (NEB). PCR amplification was performed using Q5 high-fidelity DNA polymerase (NEB) or GoTaq DNA polymerase (Promega). PCR fragments were purified using ZYMO Fragment Recovery Kit (ZYMO research). Plasmids were then constructed from the purified PCR fragments using Gibson Assembly kit (NEB) and transformed into chemically competent E. coli cells by heat shock and extracted using QIAprep Spin Miniprep Kit (Qiagen). All procedures were performed according to the manufacturer's instructions. All engineered Y. lipolytica strains were constructed by transforming linearized plasmids (Not1 digested) using the lithium acetate method. Recombinants were verified by PCR amplification from genomic DNA. All carotenoid biosynthetic genes evaluated in this study were codon-optimized for Y. lipolytica.

[0152] Disruption of TRP1 in the po1f strain using CRISPR-Cas9 For TRP1 disruption, a CRISPR-Cas9 plasmid (Schwartz et al., 2016) containing gRNA (ACGCCGAGGAGTGGTACCGG) (SEQ ID NO: 30) targeting the TRP1 (YALI0B07667g) gene of Y. lipolytica was transformed into the po1f strain using Ura3 as an auxotrophic marker. Strains with tryptophan auxotrophy were obtained by selection on YNB-Ura and YNB-Ura-Trp plates. The positive clones were then inoculated onto YPD plates and subcultured three times to lose the CRISPR-Cas9 plasmid and become the po1f-T strain (ura3 - , leu2 - , trp1 -) was obtained.

[0153] Curation of auxotrophic markers using the Cre-loxP system Plasmid pYLMA-Cre was transformed into the target Y. lipolytica strain to rescue the URA3, LEU2, and TRP1 markers. Transformants were selected on YPD agar plates supplemented with hygromycin B at a final concentration of 250 mg / L (Sigma-Aldrich). After 2–3 days of culture, colonies were transferred to new YPD plates containing hygromycin B for another day to ensure further successful marker removal. Marker curation was confirmed by subculturing colonies on YNB-Ura, YNB-Leu, and YNB-Trp agar plates, respectively. Successful removal of all three markers results in a phenotype exhibiting uracil, leucine, and tryptophan deficiency. The positive strains were then incubated on YPD agar plates at 30 °C for 24 h in two to three replicates to remove the plasmid pYLMA-Cre in the cells.

[0154] Shake flask fermentation A single colony of the recombinant strain was picked from the plate, inoculated into 2 mL of YPD medium, and grown overnight (16–18 h) at 30 °C and 230 rpm. The culture was then transferred to 10 mL of YPD medium (initial OD 600 = 0.1) and cultured at 30 °C with shaking at 230 rpm for 3–5 days. When applicable, 30 mM isoprenol (Sigma-Aldrich) was added to the YPD medium when the glucose in the culture was nearly consumed.

[0155] Bioreactor Fermentation Fed-batch fermentations were performed in a 3 L bioreactor (New Brunswick Bioflo115 system). The first fermentation was completed with 1 L of medium containing 100 g / L glucose, 100 g / L peptone, and 50 g / L yeast extract. The temperature was maintained at 30°C. Dissolved oxygen was controlled at 20% with an agitation cascade at 250-800 rpm. Air was injected into the fermenter at 2 vvm. The pH was maintained at 6.8 by feeding 5 M NaOH or 5 M HCL. Foaming was prevented by the addition of antifoam 204 (Sigma-Aldrich). The fed-batch process was performed in a 10 x Y medium consisting of 100 g / L yeast extract, 100 g / L peptone, and 500 g / L glucose. 10 P 10 D 50 The medium was fed after 48 hours of incubation. Once the medium feeding was started, agitation and aeration were changed and kept constant at 600 rpm and 0.3 vvm, respectively. Samples were taken every 24 hours and the OD 600 , glucose concentration, and carotenoid titer were measured.

[0156] Quantification of residual glucose in the medium For quantification of residual glucose, 500 μL samples were extracted from the cultures. Cells were centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a 0.2 μm syringe filter before being injected into an Agilent technologies 1260 high performance liquid chromatography (HPLC) equipped with a refractive index detector. A Bio-Rad HPX-87H column was used for separation, with 14 mM sulfuric acid as the mobile phase at a rate of 0.7 mL / min. The injection volume was 10 μL. The column temperature was 50 °C.

[0157] Lipid extraction and quantification Fatty acids synthesized by Y. lipolytica (palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2)) were quantified using gas chromatography coupled to a flame ionization detector (GC-FID). 0.1–1 mL of cell culture was extracted from each bioreactor, ensuring that the samples contained approximately 1 mg of biomass. A centrifugation step was performed at 16,000 g for 10 min and the supernatant was discarded. 0.5 mL of 0.5 M sodium hydroxide-methanol solution (20 g / L sodium hydroxide in anhydrous methanol) was mixed with the cell pellet and 100 µL of internal standards containing 2 mg / mL methyl tridecanoate (Sigma-Aldrich) and 2 mg / mL glyceryl triheptadecanoate (Sigma-Aldrich) dissolved in hexane were added. Methyl tridecanoate was used to correct for volume loss during sample preparation, and glyceryl triheptadecanoate was used to correct for transesterification efficiency. The samples were vortexed for 1 h to transesterify lipids to fatty acid methyl esters (FAMEs). Afterwards, 40 μL of 98% sulfuric acid (Sigma-Aldrich) was added to neutralize the pH. Next, 0.5 mL of hexane was added to extract FAMEs, followed by vortexing for 30 min. Afterwards, the mixture was centrifuged at 12,000 g for 1 min to remove cell debris, and the top hexane layer was extracted and analyzed. Separation of FAME species was performed on an Agilent HP-INNOWax capillary column. The injection volume was 1 μL, the split ratio was 10, and the injection temperature was 260 °C. The column was kept at a constant temperature of 200 °C and helium was used as the carrier gas at a flow rate of 1.5 mL / min. The FID was set at a temperature of 260° C., with helium make-up gas, hydrogen, and air flow rates of 25 mL / min, 30 mL / min, and 300 mL / min, respectively.

[0158] Extraction and quantification of intracellular metabolites To extract intracellular metabolites (e.g., IPP / DMAPP, and GGPP), 1 mL of culture was filtered through a 25 mm 0.2 μm nylon filter by vacuum filtration. The cells were immediately washed with 2 mL of water preheated to 30 °C, and the filter was immersed in ice-cold extraction buffer (40% methanol + 40% acetonitrile + 20% water). After 20 min of incubation at -20 °C, the extract was centrifuged at 16,000 rpm for 10 min, and the supernatant was transferred to a new tube and dried. The sample was resuspended in 50 μL of water and then centrifuged at 16,000 rpm for 10 min. Metabolites in the supernatant were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) consisting of an Agilent 1100 series LC system and an AB Sciex API-4000 MS. 10 μL of sample was injected and separation was performed on a Waters XBridge C-18 column with a mobile phase consisting of solution A (0.1% tributylamine, 0.12% acetic acid, 0.5% of 5 M NH4OH in water, v / v) and solution B (100% acetonitrile). The flow rate was 0.3 mL / min and the following gradient was used: 0–5 min, 0% B; 5–20 min, 0–65% B; 20–25 min, 65% B; 25–30 min, 100% B; 30–35 min, 100% B; 35–36 min, 100–0% B; 0% B until 45 min. Analytes were then compared to a calibration curve prepared using chemical standards purchased from Sigma-Aldrich and Cayman Chemicals.

[0159] Labeling experiments The strains used for labeling studies were grown in YNB medium containing [U- 13[C] glucose was reinstated as the sole carbon source. These were then subcultured in the same medium and grown at 30 °C until early stationary phase. Samples were taken using the same intercellular metabolite extraction method before starting the pulse addition of additional carbon sources. 10 mM stearic acid was then added to the corresponding cultures and measurements of metabolite isotopic enrichment were performed at different time points. The optical density associated with each sample was also recorded. IPP / DMAPP and GGPP were quantified by LC-MS / MS as previously described. All MS data obtained from the labeling experiments were corrected using IsoCor for natural abundance (Millard et al., 2012).

[0160] Carotenoid Extraction Carotenoid extraction was performed as described (Asker et al., 2017) with the following modifications. Briefly, 100 μL of culture was centrifuged at 16,000 g for 1 min and the cell pellet was suspended in 900 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich) and then heated at 50 °C for 1 h in a water bath until the cells were bleached. The DMSO extract was briefly mixed with 450 μL of methanol and centrifuged at 16,000 g for 5 min. The resulting supernatant was transferred to a 96-well assay plate or glass vials for carotenoid analysis and quantification.

[0161] Carotenoid Analysis and Quantification Carotenoid production was expressed as grams per liter of fermentation broth (g / L) and milligrams per gram of dry cell weight (mg / g DCW). Optical density was measured at 600 nm using a Thermo Spectronic Genesys 20 (Thermo Scientific) and used to calculate cell mass (DCW = 0.35 × OD for β-carotene). 600 , DCW = 0.30 x OD for lycopene 600, Figures 40A-40B). Analysis and quantification of β-carotene was performed by HPLC (SHIMADZU LC-20 AT) equipped with a Kromasil C18 column (4.6 mm x 250 mm) and UV / VIS detection at 450 nm. The mobile phase consisted of acetonitrile-methanol-isopropanol (5:3:2 v / v) with a flow rate of 1 mL / min at 40 °C. Analysis and quantification of lycopene was performed by Spectramax M2eMicroplate Reader (Molecular devices) or HPLC at 470 nm. Standard curves of β-carotene and lycopene (Sigma-Aldrich) were prepared by running the same extraction process as the samples.

[0162] Quantitative real-time PCR Real-time PCR (RT-PCR) was used to estimate relative gene expression. mRNA extracted with MasterPure™ Yeast RNA Purification Kit (Lucigen, Wisconsin, USA) was used as a template. RT-PCR was performed using the iScript™ One-Step RT-PCR Kit and SYBR Green Supermix (Bio-Rad, USA) on an iCycler (Bio-Rad, USA) according to the manufacturer's instructions. ACT1 was used as an internal control gene for normalization. Relative gene expression was estimated using comparative 2 -△△CT OR 2 -△CT The calculation was performed using the method.

[0163] In vitro enzyme assay Yeast microsomes for in vitro enzyme assays were prepared as previously described ( Pompon et al., 1996 ). Briefly, strains harboring wild-type or mutant CarRP were grown overnight at 30°C in YNB medium and then inoculated into 200 mL of YNB medium to reach initial OD . 600The pH was adjusted to 0.1. After 24 hours of culture, the cells were harvested by centrifugation at 4,000 rpm for 10 minutes. The cells were then resuspended in TEK buffer (50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.1 M KCl) and the solution was kept at room temperature for 5 minutes. The cells were then harvested by centrifugation, washed with TES buffer (50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.6 M sorbitol), resuspended in TESM buffer (50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.6 M sorbitol, 14 mM 2-mercaptoethanol) and left at room temperature for 10 minutes. The cells were then harvested again by centrifugation, washed with extraction buffer (50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 1 mM PMSF) and resuspended in extraction buffer. Glass beads were added to each sample, vortexed intermittently for 30 seconds, and placed on ice for 30 seconds, which was repeated a total of 15 times. The cell pellet was then centrifuged at 4,000 rpm at 4 °C for 10 minutes, discarded, and the supernatant transferred to a 50 mL tube. The crude yeast microsomal fraction collected above was used for the in vitro assay of lycopene cyclase. Standard enzyme assays were performed in a total volume of 200 μL containing 50 mM Tris-HCl (pH 6.8), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mg of microsomal protein. Serial concentrations of lycopene (50–350 μmol / L) dissolved in dimethyl sulfoxide (DMSO) were used as substrate. The reaction was initiated by the addition of substrate and incubated at 30 °C for 16 hours with gentle shaking, then stopped by the addition of 200 μL of ethyl acetate. The solution was vortexed for 10 min and the organic phase was collected by centrifugation and analyzed by HPLC.

[0164] Generation of protein variants The variants were generated by analyzing the amino acid conservation and coevolution information of this protein family from the Position Specific Scoring Matrix (PSSM). Here, the matrix was generated using psiblast in ncbi-blast-2.7.1+ (Altschul et al., 1997) with uniref90 (Suzek et al., 2007) as the database, with an E-value of 0.01 and three iterations. For every position in the protein, the PSSM score, which represents the conservation of the amino acid, was calculated for both lycopene cyclase and other homologous proteins in this family. A higher score indicates that the amino acid at this position is more conserved. Based on the PSSM score, substitutions that could be replaced with more conserved amino acids were screened. The difference value between the potential substitution and the wild-type amino acid was calculated and the scores were sorted. All glycine substitutions were removed from the scoring. The top 25 scoring substitutions were randomly combined with double substitutions. Distance matrices were calculated using the PAM30 substitution matrix and clustered into 25 clusters using Agglomerative in sklearn (Pedregosa et al., 2011) to minimize the number of trials. Variants were randomly selected within each cluster. Scoring substitutions with ranks 26–50 were ordered as single mutation variants.

[0165] A homology model of lycopene cyclase. Using the TrRosetta server ( Yang et al., 2020 ), we submitted the sequence of the R domain (amino acids 1–239) of CarRP and generated a homology model of lycopene cyclase.

[0166] Calculation of C / N ratio in medium The C / N ratio was calculated by referring to the composition of yeast extract (Bacto™) and peptone (Bacto™) in the BD Bionutrients™ technical manual (Table 10) (legacy.bd.com / ds / technicalCenter / misc / lcn01558-bionutrients-manual.pdf). The carbon in yeast extract and peptone was ignored because it was extremely low compared to the glucose concentration. The total nitrogen in yeast extract and peptone was 10.9% and 15.4%, respectively. The C / N ratio was calculated by the following formula: X, Y, and Z represent the concentrations of glucose, yeast extract, and peptone, respectively.

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[0167] [Table 6]

[0168] [Table 7-1] [Table 7-2]

[0169] [Table 8]

[0170] [Table 9-1] [Table 9-2] [Table 9-3]

[0171] [Table 10]

[0172] References:

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Claims

1. Genetically modified yeast cells (engineered cells), including: (i) a heterologous gene, wherein the heterologous gene encodes an enzyme having β-galactosidase (LacA) activity; (ii) one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate; (iii) one or more heterologous genes encoding one or more enzymes of the Leloir pathway; and (iv) one or more heterologous genes encoding one or more enzymes of the mevalonate pathway.

2. 10. The modified cell of claim 1, wherein the modified cell is an oleaginous yeast cell.

3. 3. The modified cell of claim 2, wherein the oleaginous cell is a Yarrowia lipolytica cell.

4. 4. The modified cell of any one of claims 1 to 3, wherein the one or more heterologous genes encoding one or more enzymes capable of converting lactate to pyruvate are selected from the group consisting of lactate transporter (JEN1) and lactate dehydrogenase (LDH).

5. 4. The modified cell of any one of claims 1 to 3, wherein the one or more heterologous genes encoding one or more enzymes of the Leloir pathway are selected from the group consisting of GAL10M, GAL1, GAL7, and GAL10E.

6. 4. The modified cell of any one of claims 1 to 3, wherein the one or more heterologous genes encoding one or more enzymes of the mevalonate pathway are selected from the group consisting of GGPPS, CarRP, and CarB.

7. The modified cell of claim 6, wherein the GGPPS is GGPPSxd from Xanthophyllomyces dendrorhous, GGPPSsa from Sulfolobus acidocaldarius, GGPPStc from Taxus canadensis, GGPPSpa from Pantoea agglomerans, or GGPPSyl from Yarrowia lipolytica.

8. The modified cell of any one of claims 1 to 3, further comprising a heterologous gene encoding an enzyme having lycopene β-cyclase activity.

9. The modified cell of claim 8, wherein the enzyme having lycopene β-cyclase activity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:

1.

10. The modified cell of claim 9, wherein the enzyme having lycopene β-cyclase activity comprises an amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.

11. The modified cell of claim 10, further comprising heterologous genes encoding tHMGR, ERG12, IDI, and ERG20 of the mevalonate (MVA) pathway, and / or choline kinase (CK) and isopentenyl phosphate kinase (IPK).

12. The modified cell of any one of claims 1 to 3, further comprising: (i) a heterologous gene encoding an enzyme having β-carotene ketolase (CrtW) activity; and (ii) a heterologous gene encoding an enzyme with β-carotene hydroxylase (CrtZ) activity;

13. The modified cell of claim 12, wherein an enzyme having CrtW activity is fused to an enzyme having CrtZ activity (CrtW / CrtZ fusion enzyme).

14. The modified cell of claim 13, wherein the CrtW / CrtZ fusion enzyme comprises a localization signal.

15. The modified cell of claim 14, wherein the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisomes, and / or fat bodies.

16. The modified cell of any one of claims 1 to 3, further comprising a heterologous gene encoding an enzyme having lycopene beta cyclase activity and / or a heterologous gene encoding an enzyme having lycopene epsilon cyclase activity.

17. The modified cell of claim 16, wherein the enzyme having lycopene β-cyclase activity comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO:

1.

18. 17. The modified cell of claim 16, further comprising a heterologous gene encoding an enzyme having carotenoid hydroxylase 1 (LUT1) activity and / or a heterologous gene encoding an enzyme having carotenoid hydroxylase 5 (LUT5) activity.

19. Genetically modified yeast cells (engineered cells), including: (i) a first heterologous gene, wherein the first heterologous gene encodes an enzyme having β-carotene ketolase (CrtW) activity; and (ii) a second heterologous gene, wherein the second heterologous gene encodes an enzyme having β-carotene hydroxylase (CrtZ) activity; The modified cells now produce β-carotene.

20. 20. The modified cell of claim 19, wherein the modified cell is an oleaginous yeast cell.

21. 21. The modified cell of claim 20, wherein the oleaginous cell is a Yarrowia lipolytica cell.

22. 22. The modified cell of any one of claims 19 to 21, wherein the enzyme having CrtW activity is fused to an enzyme having CrtZ activity.

23. 23. The modified cell of claim 22, wherein the CrtW / CrtZ fusion enzyme comprises a localization signal.

24. 24. The modified cell of claim 23, wherein the localization signal targets the CrtW / CrtZ fusion enzyme to the endoplasmic reticulum, peroxisomes, and / or fat bodies.

25. A method for converting a carbon source into lycopene and / or beta-carotene, comprising: (i) contacting the modified cell of any one of claims 1 to 3 with a carbon source; and (ii) incubating the modified cells with a carbon source for a sufficient time to convert the carbon source into lycopene and / or β-carotene;

26. 26. The method of claim 25, wherein the carbon source is acid whey.

27. 26. The method of claim 25, wherein the carbon source is converted to lycopene.

28. 26. The method of claim 25, wherein the carbon source is converted to β-carotene.

29. A method for converting a carbon source into astaxanthin, including: (i) contacting the modified cell of claim 12 with a carbon source; and (ii) incubating the modified cells with a carbon source for a sufficient time to convert the carbon source to astaxanthin;

30. 30. The method of claim 29, wherein the carbon source is acid whey.

31. A method for converting a carbon source to alpha-carotene, comprising: (i) contacting the modified cell of any one of claims 1 to 3 with a carbon source; and (ii) incubating the modified cells with a carbon source for a sufficient time to convert the carbon source to α-carotene;

32. 32. The method of claim 31 , wherein the carbon source is acid whey.

33. Methods for converting carbon sources into lutein, including: (i) contacting the modified cell of any one of claims 1 to 3 with a carbon source; and (ii) incubating the modified cells with a carbon source for a sufficient time to convert the carbon source into lutein;

34. 34. The method of claim 33, wherein the carbon source is acid whey.

35. An enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO:

2.

36. An enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO:

3.

37. An enzyme having lycopene β-cyclase activity, comprising the amino acid sequence represented by SEQ ID NO: 4.

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