Yeast strains with high protein content

Abstract

The present invention relates to yeast strains and uses thereof. In particular, the present invention relates to yeast strains capable of expressing proteins at high levels when grown on industrial waste, as well as the use of such strains in the preparation of animal feed.

Description

Technical Field

[0001] This application claims priority to Australian Provisional Patent Application No. 2023901560 (filed on 19 May 2023), the entire contents of which are incorporated herein by reference.

[0002] This invention relates to yeast strains and their uses. In particular, this invention relates to yeast strains that can express proteins at high levels when grown on industrial waste, and the use of such strains in the preparation of animal feed. Background Technology

[0003] Any discussion of prior art throughout this specification should not be construed as an admission that such prior art is widely known or constitutes part of common general knowledge in the art.

[0004] Yeasts of the genus *Saccharomyces* are widely used in animal feed, serving as both probiotics and as a source of easily digestible proteins and nucleic acids.

[0005] Yeast used in animal feed production needs to possess several characteristics to ensure cost-effective production of high-protein animal feed. These characteristics include high growth rate, high yield, efficient nitrogen utilization, and the ability to achieve high protein levels when grown in fed-batch cultures. In these fed-batch processes, substrate costs account for a significant portion of manufacturing costs. Therefore, the listed characteristics have a significant impact on the feasibility of industrial processes.

[0006] Currently, the types of *Saccharomyces cerevisiae* strains used for animal feed production are limited in terms of the economically viable carbon sources for growth substrates. Most yeast biomass produced globally is grown in fed-batch cultures using molasses (with sucrose as the sugar source) or corn syrup (with glucose as the sugar source). While molasses is a byproduct of the sugar industry, it has significant value as a substrate for animal feed and many industrial processes requiring the production of microbial biomass. Therefore, there is likely to be substantial demand for molasses, and its price may be affected by rising costs, impacting the economic viability of producing *Saccharomyces cerevisiae* as a protein source. Similarly, corn syrup is widely used in various human food products.

[0007] While strains currently used in the animal feed industry are well-suited for biomass production, there is a growing demand for yeast strains that can efficiently utilize carbon sources other than sucrose or glucose while producing high-protein yeasts suitable for animal feed. For example, biodiesel and corn ethanol production both generate byproducts with high glycerol content. Furthermore, pulp manufacturing using sulfite processes produces significant amounts of waste rich in xylose and acetic acid.

[0008] In cases where glycerol is used as a substrate for yeast biomass production, it has been widely reported that yeasts of the genus *Saccharomyces* can grow slowly on glycerol if additional nutrients (such as yeast extract) are provided. Adding yeast extract in the industrial process of yeast biomass production is extremely expensive; therefore, it would be advantageous to develop *Saccharomyces* strains that can utilize glycerol in high yields and at high productivity without the addition of expensive nutrients such as yeast extract.

[0009] Regarding the use of xylose as a substrate for yeast biomass production, it has been widely reported that yeasts in the genus *Saccharomyces* have an extremely poor ability to utilize xylose, and therefore it has been believed for many years that *Saccharomyces* is completely incapable of utilizing xylose.

[0010] Therefore, it would be advantageous to develop novel yeast strains that can grow efficiently on a variety of alternative carbon sources (such as xylose, glycerol, acetate, lactic acid, and ethanol) while producing high levels of protein under suboptimal growth and protein production conditions.

[0011] Therefore, the object of the present invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative. Summary of the Invention

[0012] The inventors have developed a non-genetically engineered high-protein yeast strain (Saccharomyces) that grows efficiently on industrial waste substrates containing alternative carbon sources such as glycerol, xylose, acetate, lactic acid, and ethanol, and is suitable for animal feed.

[0013] A first aspect of the present invention provides a yeast strain comprising the following characteristics:

[0014] (i) Under the same conditions, it can utilize industrial waste to grow with approximately the same efficiency as the yeast strain (strain MBG 4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171; and

[0015] (ii) When grown under the same conditions, it contains approximately the same protein content as strain MBG 4917.

[0016] A second aspect of the present invention provides a strain selected from the genus *Saccharomyces*:

[0017] (a) A yeast strain (strain MBG4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171; and

[0018] (b) Derivatives of strain MBG 4917.

[0019] A third aspect of the present invention provides a yeast strain capable of growing on industrial waste, and having a protein content of at least 50% by weight.

[0020] In one embodiment, the industrial waste is selected from the group consisting of corn ethanolstillage, palm kernel hydrolysis products, and glycerol derived from biodiesel.

[0021] A fourth aspect of the present invention provides a yeast strain capable of growing using xylose, glycerol, acetate, lactic acid and / or ethanol, and having a protein content of at least 50% by weight.

[0022] In one embodiment, the protein content of the yeast strain is in the range of about 50% to about 70% by weight.

[0023] In one embodiment, the protein content of the yeast strain is at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0024] In one embodiment, the protein content of the yeast strain is approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, or approximately 69% by weight.

[0025] In one embodiment, the yeast strain is a non-genetically modified yeast strain.

[0026] In one embodiment, the yeast strain is a strain (strain MBG 4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171.

[0027] The fifth aspect of the invention provides a yeast strain (strain MBG 4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171.

[0028] A sixth aspect of the present invention provides a method for producing yeast with a protein content of at least 50% by weight, the method comprising: culturing a yeast strain according to any one of the first to fifth aspects together with a substrate composed of industrial waste under conditions that promote the growth of the yeast strain.

[0029] In one embodiment, the industrial waste is selected from the group consisting of corn ethanolstillage, palm kernel hydrolysis products, and glycerol derived from biodiesel.

[0030] A seventh aspect of the present invention provides a method for producing yeast with a protein content of at least 50% by weight, the method comprising: culturing a yeast strain according to any one of the first to fifth aspects together with a substrate comprising xylose, glycerol, acetate, lactic acid and / or ethanol under conditions that promote the growth of the yeast strain.

[0031] In one embodiment, the protein content of the yeast is in the range of about 50% to about 70% by weight.

[0032] In one embodiment, the yeast contains at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0033] In one embodiment, the yeast contains about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% by weight.

[0034] In one embodiment, the yeast strain is able to grow using xylose.

[0035] In one embodiment, the yeast strain is able to grow using xylose as the sole carbon source.

[0036] In one embodiment, the yeast strain grows on xylose at a rate at least 100 times faster than EthanolRed at 48 hours.

[0037] In one embodiment, under test T1 conditions, the yeast strain grows on xylose at a rate at least 100 times faster than Ethanol Red at 48 hours.

[0038] In one embodiment, under test T1 conditions, the yeast strain grew on xylose approximately 130 times faster than Ethanol Red at 48 hours.

[0039] In one embodiment, the yeast strain is able to grow using glycerol.

[0040] In one embodiment, the yeast strain is able to grow using glycerol as the sole carbon source.

[0041] In one embodiment, the yeast strain grows on glycerol at a rate at least 50 times faster than EthanolRed at 48 hours.

[0042] In one embodiment, under test T2 conditions, the yeast strain grew at least 50 times faster on glycerol than Ethanol Red at 48 hours.

[0043] In one embodiment, under test T2 conditions, the yeast strain grew on glycerol approximately 58 times faster than Ethanol Red at 48 hours.

[0044] In one embodiment, the yeast strain is capable of growing using acetate.

[0045] In one embodiment, the yeast strain is able to grow using acetate as the sole carbon source.

[0046] In one embodiment, the yeast strain grows on acetate at a rate at least 15 times faster than Ethanol Red at 48 hours.

[0047] In one embodiment, under test T3 conditions, the yeast strain grew at least 15 times faster on acetate than Ethanol Red at 48 hours.

[0048] In one embodiment, under test T3 conditions, the yeast strain grew on acetate approximately 19 times faster than Ethanol Red at 48 hours.

[0049] In one embodiment, the yeast strain is able to utilize lactic acid for growth.

[0050] In one embodiment, the yeast strain is able to grow using lactic acid as the sole carbon source.

[0051] In one embodiment, the yeast strain grows on lactic acid at a rate at least 5 times faster than EthanolRed at 48 hours.

[0052] In one embodiment, under test T4 conditions, the yeast strain grows on lactic acid at a rate at least 5 times faster than Ethanol Red at 48 hours.

[0053] In one embodiment, under test T4 conditions, the yeast strain grew on lactic acid approximately 7 times faster than Ethanol Red at 48 hours.

[0054] In one embodiment, the yeast strain is capable of growth using ethanol.

[0055] In one embodiment, the yeast strain is able to grow using ethanol as the sole carbon source.

[0056] In one embodiment, the yeast strain grows at least 5 times faster on ethanol than EthanolRed at 48 hours.

[0057] In one embodiment, under test T5 conditions, the yeast strain grows at least 5 times faster on ethanol than Ethanol Red at 48 hours.

[0058] In one embodiment, under test T5 conditions, the yeast strain grew approximately 7 times faster on ethanol than Ethanol Red after 48 hours.

[0059] In one embodiment, the yeast strain is capable of growing on corn ethanol distillation waste liquid.

[0060] The eighth aspect of the invention provides a yeast produced by the method according to the sixth or seventh aspect, having a protein content of at least 50% by weight.

[0061] In one embodiment, the protein content of the yeast is in the range of about 50% to about 70% by weight.

[0062] In one embodiment, the yeast contains at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0063] In one embodiment, the yeast contains about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% by weight.

[0064] A ninth aspect of the present invention provides an animal feed composition comprising a yeast strain according to any one of the first to fifth aspects or a yeast according to the eighth aspect.

[0065] The tenth aspect of the present invention provides a method for producing yeast with a protein content of at least 50% by weight, the method comprising: culturing a yeast strain according to any one of the first to fifth aspects together with corn ethanol distillation waste liquid under conditions that promote the growth of the yeast strain.

[0066] In one embodiment, the protein content of the yeast is in the range of about 50% to about 70% by weight.

[0067] In one embodiment, the yeast contains at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0068] In one embodiment, the yeast contains about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% by weight.

[0069] In one embodiment, the yeast strain grown on corn ethanol distillation waste liquid produces approximately 0.4 g of usable substrate per gram.

[0070] In one embodiment, the yeast strain grows at a rate of approximately 2.3 g of yeast per liter per hour on corn ethanol distillation waste liquid.

[0071] In one embodiment, the yeast strain grown on corn ethanol distillation waste liquid has a protein content of approximately 58%.

[0072] In one embodiment, the total available carbon for the yeast strain grown on corn ethanol distillation waste liquid is from about 10% w / v to about 20% w / v.

[0073] In one embodiment, the yeast strain grown on corn ethanol distillation waste liquid has a protein yield of about 0.3 g yeast per gram of total available carbon to about 0.4 g yeast per gram of total available carbon.

[0074] In one embodiment, the yeast strain grown on corn ethanol distillation waste liquid has a protein content of about 58% to about 60%.

[0075] In one embodiment, the yeast strain is capable of growing using biodiesel-derived glycerol.

[0076] The eleventh aspect of the present invention provides a method for producing yeast with a protein content of at least 50% by weight, the method comprising: culturing a yeast strain according to any one of the first to fifth aspects together with biodiesel-derived glycerol under conditions that promote the growth of the yeast strain.

[0077] In one embodiment, the protein content of the yeast is in the range of about 50% to about 70% by weight.

[0078] In one embodiment, the yeast contains at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0079] In one embodiment, the yeast contains about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% by weight.

[0080] In one embodiment, the yeast strain grown on biodiesel-derived glycerol produces approximately 0.4 g of usable substrate per gram.

[0081] In one embodiment, the yeast strain produces approximately 3.1 g of yeast per liter per hour on biodiesel-derived glycerol.

[0082] In one embodiment, the yeast strain grown on biodiesel-derived glycerol has a protein level of approximately 60%.

[0083] In one embodiment, for the yeast strain grown on biodiesel-derived glycerol / cane molasses, the total available carbon is approximately 30% w / v.

[0084] In one embodiment, the yeast strain grown on biodiesel-derived glycerol / cane molasses has a protein yield of about 0.4 g yeast per gram of total available carbon to about 0.5 g yeast per gram of total available carbon.

[0085] In one embodiment, the yeast strain grown on biodiesel-derived glycerol / cane molasses has a protein content of about 53% to about 65%.

[0086] The twelfth aspect of the present invention provides a method for producing yeast with a protein content of at least 50% by weight, the method comprising: culturing a yeast strain according to any one of the first to fifth aspects together with palm kernel hydrolysate under conditions that promote the growth of the yeast strain.

[0087] In one embodiment, the protein content of the yeast is in the range of about 50% to about 70% by weight.

[0088] In one embodiment, the yeast contains at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, or at least about 69% by weight.

[0089] In one embodiment, the yeast contains about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% by weight.

[0090] In one embodiment, the yeast strain grown on palm kernel hydrolysate produces approximately 0.45 g of yeast per gram of available substrate.

[0091] In one embodiment, the yeast strain grows at a rate of approximately 3.7 g of yeast per liter per hour on palm kernel hydrolysate.

[0092] In one embodiment, the protein level of the yeast strain grown on palm kernel hydrolysate is approximately 58%.

[0093] In one embodiment, the total available carbon for the yeast strain grown on palm kernel hydrolysate is about 10-15% w / v.

[0094] In one embodiment, the yeast strain grown on palm kernel hydrolysate has a protein yield of about 0.3 g yeast per gram of total available carbon to about 0.5 g yeast per gram of total available carbon.

[0095] In one embodiment, the yeast strain grown on palm kernel hydrolysate has a protein content of about 58% to about 65%.

[0096] In one embodiment, the derivative of strain MBG 4917 is a hybrid strain produced by culturing the first yeast strain and strain MBG 4917 under conditions that allow DNA recombination between the first yeast strain and strain MBG 4917.

[0097] In one embodiment, a derivative of strain MBG 4917 exhibits all the characteristics of strain MBG 4917.

[0098] In one embodiment, a derivative of strain MBG 4917 can be prepared by culturing the first yeast strain and strain MBG 4917 under conditions that allow DNA recombination between the first yeast strain and strain MBG 4917.

[0099] In one embodiment, culturing the first yeast strain and strain MBG 4917 under conditions that allow DNA recombination between the first yeast strain and strain MBG 4917 comprises:

[0100] (i) To induce spore production in the first yeast strain and strain MBG 4917;

[0101] (ii) The spores produced by the first yeast strain and the spores produced by strain MBG 4917 germinate and hybridize.

[0102] Methods for spore production, germination, and hybridization of yeast strains (especially those of the genus *Yeast*) are known in the art and are described, for example, in the following literature: Ausubel et al. 1997, Current Protocols in Molecular Biology, Volume 2, pages 13.2.1 to 13.2.5 (John Willey & Sons Inc); and Chapter 7, “Sporulation and Hybridisation of yeast” by RR Fowell, in “The Yeasts” vol 1, AH Rose and JS Harrison (Eds), 1969, Academic Press.

[0103] In one implementation, the yeast strain can be cultured under conditions that allow cell fusion. Methods for generating intraspecific or interspecific hybrids using cell fusion techniques are described, for example, in the following literature: Spencer et al. (1990), Yeast Technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York.

[0104] In another implementation, the yeast strain can be cultured under conditions that allow cytoduction. Methods for cytoduction are described, for example, in the following literature: Inge-Vechymov et al. (1986) Genetika 22: 2625-2636; Johnston (1990) in, Yeast technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York.

[0105] In one embodiment, the derivative of strain MBG 4917 may be a mutant of that strain. Methods for producing mutants of yeasts (particularly *Saccharomyces cerevisiae* mutants) are known in the art and are described, for example, in the following literature: Lawrence CW (1991) Methods in Enzymology, 194: 273-281.

[0106] definition

[0107] In describing and claiming this invention, the following terms have been used according to their definitions listed below. It should also be understood that the terminology used herein is for the purpose of describing embodiments of the invention only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0108] As used herein, the term "about" may refer to within one or more standard deviations according to art practice. Alternatively, "about" may refer to a range of up to 10%. When numerical values ​​are given in the specification and claims, the meaning of "about" should be assumed to be within the acceptable error range of that value.

[0109] In the context of this invention, the words “comprising,” “including,” and similar terms should be interpreted in an inclusive rather than exclusive sense, that is, in the sense of “including but not limited to.”

[0110] The terms "preferred" and "ideally" refer to technical solutions that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the statement of one or more preferred embodiments does not imply that other embodiments are useless, nor is it intended to exclude other embodiments from the scope of the invention.

[0111] Unless otherwise stated in the operational examples, all figures used herein to indicate the quantity of components or reaction conditions should be understood to be modified by the term “about” in all cases.

[0112] When using endpoints to list a range of values, the range includes all values ​​in between (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0113] As used herein, the term "industrial waste" refers to fluid and / or aqueous residues and byproducts from industrial processes, such as corn ethanol plants, biodiesel plants, pulp mills, and similar processes. In the context of this invention, the phrases "industrial waste" and "industrial waste stream" are used interchangeably.

[0114] As used in this article, the term "efficiency" in the context of yeast growth refers to the rate of yeast growth that is comparable to the rate of growth in a medium that promotes optimal or near-optimal yeast growth.

[0115] As used in this article, the terms "high protein content" or "high protein level" refer to 50% or more protein on a dry matter basis.

[0116] As used herein, the term "derivative" means a yeast that has the same or similar phenotype as the yeast of the present invention, derived from the yeast of the present invention through breeding and / or mutation, or a genetically engineered version of the yeast of the present invention that has the same or similar phenotype.

[0117] As used in this article, the term "alternative carbon source" refers to a non-carbohydrate carbon source.

[0118] As used herein, the term "non-genetically engineered" refers to yeast strains that have not been genetically modified using techniques such as gene insertion, gene knockout, gene editing, or transgenic technology. Instead, these yeast strains are developed or bred using conventional methods such as selection, hybridization, or mutation breeding to achieve desired characteristics or traits.

[0119] Brief description of the invention

[0120] The inventors recognized that it would be advantageous to grow yeast on readily available, low-cost substrates (such as byproducts of large-scale industrial processes) to produce yeast with high protein content suitable for animal feed.

[0121] Therefore, the present invention relates to a non-genetically engineered yeast strain with a high protein content suitable for addition to animal feed, which can grow on substrates currently considered unsuitable or undesirable for industrial-scale yeast production and has a sufficiently high protein content suitable for use in animal feed.

[0122] The substrates that the yeast of this invention can effectively utilize include industrial waste, such as waste from biodiesel / corn ethanol production and pulp production, which are rich in glycerol, xylose and / or acetic acid.

[0123] Current industrial processes for producing yeast with high protein content require optimal conditions, which necessitates the use of carbon sources such as molasses or corn syrup. The yeast of this invention can be grown on waste substrates and still produce protein levels equal to or higher than those of yeast conventionally grown using molasses or syrup.

[0124] Strain MBG 4917 was produced through an evolutionary and breeding program that screened for yeasts capable of growing using multiple carbon sources, including xylose, glycerol, and / or acetate, as their sole carbon source. This strain was developed entirely using methods that do not involve genetic engineering.

[0125] Strain MBG 4917 was deposited on May 17, 2023, in accordance with the Budapest Treaty with the National Measurement Institute (NMI), at 1 / 153 Bertie Street, PortMelbourne, Victoria, Australia 3207, NMI accession number V23 / 009171.

[0126] The yeast of this invention, with its high protein content, is suitable as an additive in various animal feed formulations that require a source of enhanced protein that is easily digestible and has low production costs. Therefore, the yeast of this invention can be added to animal feed formulations designed for fish, poultry, pigs, and other livestock and farm animals. Appropriate animal feed formulations are widely reported in the scientific and technical literature and are well known to those skilled in the art (see, for example, Animal Feed Formulations, Pesti & Miller, Springer Nature BV, 1993; Basic Animal Nutrition and Feeding, Pond et al., John Wiley & Sons Inc., 2004; Developing Animal Feed Products, Partheeban (Ed.), Burleigh Dodds Science Publishing Limited, 2021; Agboola et al. 2021, Reviews in Aquaculture 13: 949-970; Abasset et al. 2018, Aquaculture International 26: 843-855; Ozório et al. 2012, Animals 2: 16-24; Aghdamshahriar et al. "The effect of yeast (Saccharomyces cerevisiae) in replacement fish meal and poultry by product protein in broiler diets."). XII European Poultry Conference, Verona, Italia. 2006; Ly etal. 2017, Journal of Agricultural Science and Technology A 7: 345-49; Winkleret al. 2011, Livestock Science 137: 168-177; Maamouri & Ben Salem 2022, Veterinary Medicine and Science 8: 398-404)

[0127] Unless otherwise stated, the present invention is practiced using conventional microbiology and classical genetics. Such techniques are well known to those skilled in the art and are well described in the literature. See, for example, Sherman et al. "Methods in Yeast Genetics" (1981), Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, New York; European Patent No. EP 0511108B.

[0128] To illustrate the nature of the invention and to provide a clearer understanding of it, the following non-limiting embodiments are provided. Example

[0129] Example 1: Growth of strain MBG 4917 using xylose as the sole carbon source (test T1)

[0130] Strain MBG 4917 is the product of a breeding program designed to produce a Saccharomyces cerevisiae strain capable of efficiently growing on industrial waste substrates containing alternative carbon sources such as glycerol, xylose, acetate, lactic acid, and ethanol. This phenotype is not found in naturally occurring yeast strains.

[0131] Strain MBG 4917 was inoculated into 50 mL of broth containing 5% xylose and 1x yeast nitrogen source (YNB, Difco), and then inoculated into 250 mL Ehrlenmeyer flasks. The inoculation optical density (OD) was approximately 0.1, which is approximately 1 x 10⁻⁶ per mL. 6 YNB contains inorganic yeast nutrients and small amounts of essential vitamins. To maintain the pH within the acceptable range for the growth of Saccharomyces strains, the minimum culture medium broth was buffered with 0.3 g / 100 mL citric acid and 0.7 g / 100 mL trisodium citrate, and the pH was adjusted to pH 5 using NaOH or HCl (strain MBG 4917 cannot utilize citric acid as a carbon source). The cultures were incubated in a track shaker at 30°C at 200 rpm to ensure cell suspension and aeration. OD values ​​of the cultures were measured at 24 and 48 hours to quantify any growth.

[0132] The growth of strain MBG 4917 was compared with that of wild-type industrial strain Ethanol Red (ER, purchased from LeSaffre Yeast Corporation).

[0133] Table 1

[0134]

[0135] The results showed that strain MBG 4917 was able to grow rapidly using xylose as the sole carbon source, while Ethanol Red could not.

[0136] Example 2: Growth of strain MBG 4917 with glycerol as the sole carbon source (test T2)

[0137] Strain MBG 4917 was inoculated into 50 mL of broth containing 3% glycerol and 1x YNB, and then into 250 mL Ehrenmeyer flasks. The inoculation OD was approximately 0.1, which is approximately 1 x 10⁻⁶ per mL. 6 To maintain the pH within the acceptable range for the growth of the *Saccharomyces* strain, the minimum culture broth was buffered with 0.3 g / 100 mL citric acid and 0.7 g / 100 mL trisodium citrate, and the pH was adjusted to pH 5 using NaOH or HCl. The cultures were incubated in a track shaker at 30°C at 200 rpm to ensure cell suspension and aeration. OD values ​​of the cultures were measured at 24 and 48 hours to quantify any growth.

[0138] The growth of strain MBG 4917 was compared with that of wild-type industrial strain of Saccharomyces cerevisiae (Ethanol Red).

[0139] Table 2

[0140]

[0141] The results showed that strain MBG 4917 could grow rapidly using glycerol as the sole carbon source, while Ethanol Red could not.

[0142] Example 3: Growth of strain MBG 4917 with acetate as the sole carbon source (test T3)

[0143] Strain MBG 4917 was inoculated into 50 mL of broth containing 0.5% acetate and 1x YNB, and then into 250 mL Ehrenmeyer flasks. The inoculation OD was approximately 0.1, which is approximately 1 x 10⁻⁶ per mL. 6 To maintain the pH within the acceptable range for the growth of the *Saccharomyces* strain, the minimum culture broth was buffered with 0.3 g / 100 mL citric acid and 0.7 g / 100 mL trisodium citrate, and the pH was adjusted to pH 5 using NaOH or HCl. The cultures were incubated in a track shaker at 30°C at 200 rpm to ensure cell suspension and aeration. OD values ​​of the cultures were measured at 24 and 48 hours to quantify any growth.

[0144] The growth of strain MBG 4917 was compared with that of wild-type industrial strain of Saccharomyces cerevisiae (Ethanol Red).

[0145] Table 3

[0146]

[0147] The results showed that strain MBG 4917 was able to grow using acetate as the sole carbon source, while Ethanol Red was unable to do so.

[0148] Example 4: Growth of strain MBG 4917 with lactic acid as the sole carbon source (test T4)

[0149] Strain MBG 4917 was inoculated into 50 mL of broth containing 3% lactic acid and 1x YNB, and then into 250 mL Ehrenmeyer flasks. The inoculation OD was approximately 0.1, which is approximately 1 x 10⁻⁶ per mL. 6 To maintain the pH within the acceptable range for the growth of the *Saccharomyces* strain, the minimum culture broth was buffered with 0.3 g / 100 mL citric acid and 0.7 g / 100 mL trisodium citrate, and the pH was adjusted to pH 5 using NaOH or HCl. The cultures were incubated in a track shaker at 30°C at 200 rpm to ensure cell suspension and aeration. OD values ​​of the cultures were measured at 24 and 48 hours to quantify any growth.

[0150] The growth of strain MBG 4917 was compared with that of wild-type industrial strain of Saccharomyces cerevisiae (Ethanol Red).

[0151] Table 4

[0152]

[0153] The results showed that strain MBG 4917 was able to grow using lactic acid as the sole carbon source, while Ethanol Red was unable to do so.

[0154] Example 5: Growth of strain MBG 4917 with ethanol as the sole carbon source (test T5)

[0155] Strain MBG 4917 was inoculated into 50 mL of broth containing 0.5% ethanol, and then into 250 mL Ehrenmeyer flasks. The inoculation OD was approximately 0.1, which is approximately 1 x 10⁻⁶ per mL. 6To maintain the pH within the acceptable range for the growth of *Saccharomyces* strains, the minimum culture broth was buffered with 0.3 g / 100 mL citric acid and 0.7 g / 100 mL trisodium citrate, and the pH was adjusted to pH 5 using NaOH or HCl. The cultures were incubated in a track shaker at 30°C at 200 rpm to ensure cell suspension and aeration. OD values ​​were measured at 24 and 48 hours to quantify any growth.

[0156] The growth of strain MBG 4917 was compared with that of wild-type industrial strain of Saccharomyces cerevisiae (Ethanol Red).

[0157] Table 5

[0158]

[0159] The results showed that strain MBG 4917 could grow rapidly using ethanol as the sole carbon source, while Ethanol Red could not.

[0160] Example 6: Yeast Growth and Protein Production

[0161] Examples 1 to 5 show that MBG 4917 can grow using xylose, glycerol, acetate, lactic acid and / or ethanol as the sole carbon source, demonstrating that this strain can grow using industrial waste.

[0162] To demonstrate that strain MBG 4917 can achieve high protein levels when grown using industrial waste, a fed-batch process was used in experiments. In this process, yeast growth is maintained through continuous feeding, achieving higher cell densities compared to batch processes. The process begins by adding nutrients (nitrogen in the form of urea and phosphorus in the form of monoammonium phosphate, along with minerals and trace metals) and a small amount of substrate (feed – yeast and available carbon compounds) to the initial water before inoculation. After inoculation, the following parameters were maintained in the fermenter:

[0163] Starting volume: 350 mL

[0164] Temperature: 30℃

[0165] pH: 4.0-6.0

[0166] Airflow rate: 2 liters per minute

[0167] Feed volume: 400 mL

[0168] Feeding is initiated approximately 2-3 hours after inoculation. The feeding rate is then controlled to remain continuous and gradually increase to sustain the growth of the increasing yeast biomass. When feeding ceases, the yeast is allowed to utilize any remaining carbon compounds in the fermenter before the process is terminated. The entire process typically takes approximately 20 to 30 hours to complete.

[0169] Growth on corn ethanol distillation waste liquid

[0170] Corn ethanol plants use yeast to ferment starch in corn into ethanol. During this process, the yeast produces glycerol as a byproduct. Although the amount of glycerol produced in a typical corn ethanol fermentation process is about one-tenth of the ethanol yield, the massive scale of the US corn ethanol industry ensures that the industry generates a significant amount of glycerol as a byproduct. After removing the ethanol by distillation, the remaining "distillation waste liquid" is concentrated in a multi-effect evaporator to produce a glycerol-rich waste syrup.

[0171] Table 6

[0172]

[0173] MBG 4917 was cultured on corn ethanol waste syrup using a fed-batch process. At the end of the fed-batch process, the yeast was harvested and analyzed. MBG 4917 produced 0.38 g of yeast per gram of substrate, with a growth rate of 2.37 g of yeast per liter per hour, and a protein level of 58.2%.

[0174] Growth on crude biodiesel-derived glycerol

[0175] Biodiesel is a renewable fuel obtained through transesterification of vegetable or animal oils with alcohols such as ethanol or methanol. Due to the large production volume of biodiesel and the high cost of purifying glycerol into a form suitable for other uses, biodiesel-derived glycerol can be considered a useless byproduct. The high biochemical oxygen demand (BOD) of glycerol waste streams makes their treatment expensive; therefore, upgrading this waste stream into higher-value yeast biomass would be highly valuable.

[0176] Strain MBG 4917 was grown on waste glycerol from a biodiesel plant, derived from plants and animals, which was diluted to a concentration of 30% w / v and supplemented with standard amounts of trace elements, vitamins, and inorganic nutrients (such as monoammonium phosphate (MAP) and urea).

[0177] At the end of the fed-batch process, the yeast was harvested and analyzed. MBG 4917 produced 0.38g of yeast per gram of substrate, with a growth rate of 3.18g of yeast per liter per hour; the protein content was achieved to the target of approximately 60% by adding urea during the fed-batch process.

[0178] Growth on palm kernel hydrolysates

[0179] Palm kernels are a byproduct of the palm oil extraction industry and can be easily hydrolyzed using mannanase to produce a mixed sugar stream. MBG 4917 was grown on palm kernel hydrolysates supplemented with standard amounts of trace elements, vitamins, and inorganic nutrients such as monoammonium phosphate (MAP) and urea.

[0180] This palm kernel hydrolysate contains the following organic compounds that can be easily consumed (MBG 4917).

[0181] Table 7

[0182]

[0183] At the end of the fed-batch process, the yeast was harvested and analyzed. MBG 4917 produced 0.45g of yeast per gram of substrate, with a growth rate of 3.77g of yeast per liter per hour and a protein content of 58.6%.

[0184] Unlike other strains of Saccharomyces cerevisiae, strain MBG 4917 is a yeast that can utilize a wider range of carbon sources and can grow on minimal media without additional nutrients.

[0185] Furthermore, this strain can accumulate extremely high levels of protein when growing using these carbon sources. Therefore, it is suitable for producing high-protein yeast biomass from the following waste streams: biodiesel glycerol, glycerol-rich syrups from corn ethanol plants, and waste streams rich in xylose and acetate from the paper and pulp industry.

[0186] Example 7: Biomass yield and protein level in fed-batch yeast culture

[0187] Under fed-batch conditions, biomass is produced using corn ethanol distillation waste syrup, palm kernel hydrolysate, and a mixture of biodiesel glycerol (plant and animal sources) / cane molasses.

[0188] Strain MBG 4917 was grown in separate fermenters containing corn ethanol distillation waste syrup, palm kernel hydrolysate, and a mixture of biodiesel glycerol (plant and animal-derived feedstock) / cane molasses.

[0189] The concentrated culture medium used for fermentation experiments contained the carbon sources detailed in Table 8, and was supplemented with the minimum mineral salts, trace metals, and vitamins required for normal yeast growth (van Hoek et al., 2000 Biotechnol Bioeng. 68:517-23). ​​Total available carbon (TUC) refers to the total carbon compounds that strain MBG 4917 can utilize for growth in the culture medium.

[0190] Table 8

[0191]

[0192] Total carbon compounds in the culture medium in which strain MBG 4917 can grow were analyzed using high performance liquid chromatography (HPLC).

[0193] Table 9: TUC composition (%w / v) of corn ethanol distillation waste liquor syrup

[0194]

[0195] These corn ethanol distillation waste liquor syrups were obtained from commercial dry-milling corn ethanol facilities and represent typical corn ethanol distillation waste liquor.

[0196] Table 10: TUC composition of palm kernel hydrolysates (%w / v)

[0197]

[0198] The palm kernel meal used to produce the hydrolysate was sourced from Castlegate James, an Australian animal feed company. The palm kernel meal was hydrolyzed using mannanase to produce the mixed sugar stream shown in the table above.

[0199] Table 11: TUC composition (%w / v) of biodiesel glycerol / cane molasses mixture

[0200]

[0201] During the fed-batch process, nitrogen and phosphorus are gradually added in the form of urea and monoammonium phosphate. The internal controls of the fermenter are as follows: aeration rate of 1-2 vvm (air volume per minute per unit volume of culture medium), pH of 4 to 6, and temperature of 30°C to 32°C for 20 to 30 hours, depending on the growth rate of yeast on each substrate.

[0202] Protein level analysis of the biomass showed that strain MBG 4917 had a high protein content (see Table 12).

[0203] Table 12

[0204]

[0205] The protein content of yeast was measured using the Dumas combustion method.

[0206] The ability of strain MBG 4917 to grow yeast under scalable conditions makes it valuable for industrial applications. This example demonstrates the industrial applicability of strain MBG 4917 and its ability to grow on actual industrial waste substrates and produce high levels of protein.

Claims

1. A strain of the genus *Saccharomyces*, comprising the following characteristics: (i) Under the same conditions, it can utilize industrial waste to grow with approximately the same efficiency as the yeast strain (strain MBG 4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171; and (ii) When grown under the same conditions, it contains approximately the same protein content as strain MBG 4917.

2. A yeast strain selected from (a) A yeast strain (strain MBG4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171; and (b) Derivatives of strain MBG 4917.

3. A yeast strain capable of growing on industrial waste, and having a protein content of at least 50% by weight.

4. The yeast strain according to claim 3, wherein the industrial waste is selected from the group consisting of corn ethanol distillation waste liquid, palm kernel hydrolysis products and biodiesel-derived glycerol.

5. A yeast strain capable of growth using xylose, glycerol, acetate, lactic acid and / or ethanol, and having a protein content of at least 50% by weight.

6. The yeast strain according to any one of claims 3-5, wherein the protein content is in the range of about 50% to about 70% by weight.

7. The yeast strain according to any one of claims 1-6, wherein the yeast strain is a non-genetically modified yeast strain.

8. A yeast strain (strain MBG4917) deposited under the Budapest Treaty and with NMI accession number V23 / 009171.

9. A method for producing yeast with a protein content of at least 50% by weight, the method comprising: The yeast strain as described in any one of claims 1-8 is cultured together with a substrate composed of industrial waste under conditions that promote the growth of the yeast strain.

10. The method of claim 9, wherein the industrial waste is selected from the group consisting of corn ethanol distillation waste liquid, palm kernel hydrolysis products, and biodiesel-derived glycerol.

11. A method for producing yeast with a protein content of at least 50% by weight, the method comprising: The yeast strain as described in any one of claims 1-8 is cultured together with a substrate containing xylose, glycerol, acetate, lactic acid and / or ethanol under conditions that promote the growth of the yeast strain.

12. A yeast produced by the method of any one of claims 9-11, wherein the protein content is at least 50% by weight.

13. An animal feed composition comprising a yeast strain as described in any one of claims 1-8 or the yeast as described in claim 12.

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