WO2020089847A1

WO2020089847A1 - Process for preventing or limiting microbial contamination during continuous culture

Info

Publication number: WO2020089847A1
Application number: PCT/IB2019/059394
Authority: WO
Inventors: Emily Agnes STONEHOUSE; Kristen M. DELEAULT; Zachary LOSORDO; John Mcbride
Original assignee: Lallemand Hungary Liquidity Management Llc
Priority date: 2018-11-02
Filing date: 2019-11-01
Publication date: 2020-05-07
Also published as: BR112021008130A2

Abstract

Long term or continuous fermentation processes are susceptible to contaminating by microorganisms that can lead to decreases in efficiency. The present disclosure provides a process for limiting a microbial contamination (between 0.1-10%) during a continuous culture of a recombinant yeast host cell in a medium. The process is based on the use of a combination of a recombinant yeast host cell having a genetic modification for reducing the expression of a noxious gene capable of converting a pro-cytotoxic agent into a cytotoxic agent and a pro-cytotoxic agent.

Description

PROCESS FOR PREVENTING OR LIMITING MICROBIAL CONTAMINATION DURING

CONTINUOUS CULTURE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. provisional patent application 62/755,244 filed on November 2, 2018 and incorporated herewith in its entirety.

TECHNOLOGICAL FIELD

The present disclosure concerns processes for preventing and/or limiting and, in some instances, monitoring microbial contamination during a continuous culture, for example, during the production of biofuel by recombinant yeast host cells. The processes are based on the use of a genetically modified yeast host cells having a genetic modification for reducing the expression of a potentially noxious gene.

BACKGROUND

Continuous fermentations (such as the Brazilian fuel ethanol fermentation and/or the continuous corn fermentation) are susceptible to contaminations. For example, in Brazilian fuel ethanol fermentations, the yeasts are pitched at the beginning of the sugarcane crushing season and are continually recycled for more than 200 days. The yeasts are recycled using continuous centrifugation and acid washing to improve productivity. Wild yeast contaminants are continually entering the fermentation since the fermentation substrates (e.g., sugarcane juice and molasses) are not sterilized. In addition, the predominate Saccharomyces cerevisiae yeast strains used in the Brazilian fuel ethanol industry are highly heterozygous and are known to have genomic rearrangements which creates challenges to the traditional molecular identification methods used to monitor yeast populations (such as, for example, microsatellite and inter-delta sequence amplification, random amplified polymorphic DNA (RAPD) or karyotyping by pulse-field get electrophoresis (PFGE)).

Monitoring and limiting contamination is important in continuous fermentations from both an economic and a processing perspective. Contaminating yeast have been associated with decreased ethanol yields, flocculation and foaming. Greater than 95% of the contaminating yeasts are reported to be other Saccharomyces strains many of which have unfavorable fermentation characteristics and can lead to large productivity losses if allowed to proliferate. Less than 5% are non-Saccharomyces such as Dekkera bruxellensis, Candida krusei and Schizosaccharomyces pombe, but these strains can cause issues if left unchecked.

There is thus a need to be provided with a stable and reliable detection method for population dynamics that is not sensitive to chromosomal recombination and would allow for a more reliable way to follow a strains presence during fermentation. There is also a need for methods that would allow for limiting microbial contamination during continuous fermentation. BRIEF SUMMARY

The present disclosure concerns the use of a pro-cytotoxic agent to limit the microbial contamination of a continuous culture of a medium of a recombinant yeast host cell and/or to make a fermentation product by the recombinant yeast host cell. The recombinant yeast host cell is engineered to be incapable of metabolizing a pro-cytotoxic agent into a cytotoxic agent. In some embodiments, the pro-cytotoxic agent is used to maintain the microbial contamination between 0.1-10% (and in some embodiments, between 0.1 -1 %) of the total microbial population in the continuous culture or during fermentation.

According to a first aspect, the present disclosure provides a process for preventing or limiting a microbial contamination caused by contaminating microorganisms during a continuous culture of a recombinant yeast host cell. The process comprising culturing the recombinant yeast host cell in the presence of a pro-cytotoxic agent, wherein the recombinant yeast host cell has a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of the pro-cytotoxic agent into a cytotoxic agent. In an embodiment, the genetic modification comprises disrupting the open reading frame of the noxious gene. In another embodiment, the genetic modification comprises increasing the expression of a gene encoding an inhibitor of expression of the noxious gene. In an additional embodiment, the process comprises: (a) culturing the recombinant yeast host cell in a first medium lacking the pro-cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first medium comprises a first total microbial population and the first threshold of contaminating microorganisms is at least above about 0.1 % of the first total microbial population; (b) when the first threshold of contaminating microorganisms is reached in the first medium, culturing the recombinant yeast host cell in a second medium having the pro- cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second medium has a second total microbial population and the second threshold < 0.1 % of the second total microbial population; and (c) if or when the second threshold of contaminating microorganisms is reached in the second medium, culturing the recombinant yeast host cell in the first medium lacking the pro-cytotoxic agent. In another embodiment, the process comprises culturing the recombinant yeast host cell in the first medium when the second threshold is reached. In still another embodiment, the process comprises: (a) culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second medium comprises a second total microbial population and the second threshold of contaminating microorganisms < 0.1 % of the second total microbial population; (b) when the second threshold of contaminating microorganisms is reached in the second medium, culturing the recombinant yeast host cell in a first medium lacking the pro-cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first medium has a first total microbial population and the first threshold is above about 0.1 % of the first total microbial population; and (c) if or when the first threshold of contaminating microorganisms is exceeded in the first medium, culturing the recombinant yeast host cell in the second medium lacking the pro-cytotoxic agent. In still another embodiment, the process comprises culturing the recombinant yeast host cell in the second medium when the first threshold is exceeded. In still another embodiment, steps (a) and (b) are repeated after step (c). In yet another embodiment, the process comprises monitoring, in the first medium, the percentage of contaminating microorganisms with respect to the first total microbial population. In still another embodiment, the process comprises monitoring, in the second medium, the percentage of contaminating microorganisms with respect to the second total microbial population. In another embodiment, the monitoring is done at least once a month or at least once a week. In still another embodiment, the monitoring comprises assessing the number of colony-forming units of the contaminating microorganisms to monitor the percentage of contaminating microorganisms. In a further embodiment, the process comprises adding the pro-cytotoxic agent to the first medium to obtain the second medium. In yet a further embodiment, the process comprises refraining from adding the pro-cytotoxic agent to the second medium to obtain the first medium. In still a further embodiment, the process comprises culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent during the continuous culture. In an embodiment, the noxious gene is at least one of FCY1 , FUR1 , URA3, LYS2, LEU2, TRP1 , HISS, MET15 or ADE2. In still another embodiment, the noxious gene comprises FCY1 and, in yet a further embodiment, the pro-cytotoxic agent is 5- fluorocytosine (5-FC). In a further embodiment, the noxious gene is FUR1 and, in yet a further embodiment, the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). In a further embodiment, the noxious gene comprises both FCY1 and FUR1 and, in yet a further embodiment, the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). In an embodiment, the concentration of 5-FC or 5-FU is between about 0.1 and about 500 ppm in the second medium. In still another embodiment, the process comprises contacting the pro- cytotoxic agent with the recombinant yeast host cell at least once, twice or thrice a year. In a further embodiment, the contaminating microorganisms comprise yeasts. In another embodiment, the recombinant yeast host cell is from Saccharomyces sp. and in further embodiment, the recombinant yeast host cell is from Saccharomyces cerevisiae.

According to a second aspect, the present disclosure provides a process for making a fermentation product from a first and/or a second fermentation medium comprising a carbohydrate. The process comprising culturing a recombinant yeast host cell in the presence of a pro-cytotoxic agent under conditions so as to allow making the fermentation product, wherein the recombinant yeast host cell has a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of the pro-cytotoxic agent into a cytotoxic agent. In an embodiment, the genetic modification comprises disrupting the open reading frame of the noxious gene. In another embodiment, the genetic modification comprises increasing the expression of a gene encoding an inhibitor of expression of the noxious gene. In an embodiment, the process comprises: (a) contacting the recombinant host cell with the first fermentation medium comprising a carbohydrate and lacking the pro-cytotoxic agent under conditions to promote the production of the fermentation product and until a first threshold of contaminating microorganisms is exceeded, wherein the first fermentation medium comprises a first total microbial population and the first threshold of contaminating microorganisms is above about 0.1 % of the first total microbial population; (b) when the first threshold of contaminating microorganisms is exceeded in the first fermentation medium, culturing the recombinant yeast host cell in the second fermentation medium having the carbohydrate and the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second fermentation medium has a second total microbial population and the second threshold < 0.1 % of the second total microbial population; and (c) if or when the second threshold of contaminating microorganisms is reached in the second fermentation medium, culturing the recombinant yeast host cell in the first fermentation medium. In an embodiment, the process comprises culturing the recombinant yeast host cell in the first fermentation medium when the second threshold is reached. In still another embodiment, the process comprises: (a) culturing the recombinant yeast host cell in a second fermentation medium having a carbohydrate and the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second fermentation medium comprises a second total microbial population and the second threshold of contaminating microorganisms < 0.1 % of the second total microbial population; (b) when the second threshold of contaminating microorganisms is reached in the second fermentation medium, culturing the recombinant yeast host cell in a first fermentation medium comprising the carbohydrate and lacking the pro-cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first fermentation medium has a first total microbial population and the first threshold is between about 1-10% of the first total microbial population; and (c) if or when the first threshold of contaminating microorganisms is exceeded in the first fermentation medium, culturing the recombinant yeast host cell in the second fermentation medium. In another embodiment, the process comprises culturing the recombinant yeast host cell in the second fermentation medium when the first threshold is exceeded. In still another embodiment, steps (a) and (b) are repeated after step (c). In still another embodiment, the process comprises monitoring, in the first fermentation medium, the percentage of contaminating microorganisms with respect to the first total microbial population. In another embodiment, the process comprises monitoring, in the second fermentation medium, the percentage of contaminating microorganisms with respect to the second total microbial population. In a further embodiment, the monitoring is done at least once a month or at least once a week. In still another embodiment, the monitoring comprises assessing the number of colony-forming units of the contaminating microorganisms to monitor the percentage of contaminating microorganisms. In a further embodiment, the process comprises adding the pro- cytotoxic agent to the first fermentation medium to obtain the second fermentation medium. In another embodiment, the process comprises refraining from adding the pro-cytotoxic agent to the second fermentation medium to obtain the first fermentation medium. In still another embodiment, the process comprises culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent during the continuous culture. In an embodiment, the noxious gene is at least one of FCY1 , FUR1 , URA3, LYS2, LEU2, TRP1 , HISS, MET15 or ADE2. In still another embodiment, the noxious gene comprises FCY1 and, in yet another embodiment, the pro-cytotoxic agent is 5-fluorocytosine (5-FC). In still another embodiment, the noxious gene is FUR1 and, in still another embodiment, the pro-cytotoxic agent is 5- fluorocytosine (5-FC) or 5-fluorouracil (5-FU). In still another embodiment, the noxious gene comprises both FCY1 and FUR1 and, in still another embodiment, the pro-cytotoxic agent is 5- fluorocytosine (5-FC) or 5-fluorouracil (5-FU). In yet another embodiment, the second fermentation medium comprises between about 0.1 and about 500 ppm of 5-FC or 5-FU. In still a further embodiment, the process comprises contacting the pro-cytotoxic agent with the recombinant yeast host cell at least once, twice or thrice a year. In an embodiment, the contaminating microorganisms comprise yeasts. In yet another embodiment, the carbohydrate is a sugarcane juice, a sugarcane derivative, corn, a corn derivative, molasses and/or a molasses derivative. In yet a further embodiment, the fermentation product is ethanol, isopropanol, n-propanol, 1 -butanol, methanol, acetone, 1 , 2 propanediol or an heterologous polypeptide. In an embodiment, the process further comprises contacting the procytotoxic agent with the recombinant yeast host cell prior to culturing the recombinant yeast host cell. In another embodiment, the process further comprises at least one fermentation cycle comprising acid washing the recombinant yeast host cell present in the first and/or second fermentation medium to obtain an acid washed recombinant microbial host cell and contacting the acid washed recombinant yeast host cell with the first and/or the second fermentation medium to promote the production of the fermentation product. In yet another embodiment, the process further comprises at least two or more fermentation cycles. In yet another embodiment, steps (a), (b) and (c) are performed at least once prior to acid washing. In still a further embodiment, steps (a), (b) and (c) are performed at least once in each fermentation cycle. In an embodiment, the genetic modification of the noxious gene does not substantially alter the fermentation performance of the recombinant yeast host cell when compared to the fermentation performance of a corresponding yeast host cell lacking the genetic modification. In yet another embodiment, the recombinant yeast host cell is from Saccharomyces sp. and in some additional embodiments, the recombinant yeast host cell is from Saccharomyces cerevisiae.

According to a third aspect, the present disclosure provides a method of determining the presence of contaminating microorganisms in a specimen having a total microbial population and comprising a recombinant yeast host cell, the recombinant yeast host cell having a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of a pro-cytotoxic agent into a cytotoxic agent. The method comprises: (a) culturing a first sample of the specimen in a selective medium comprising the pro-cytototoxic agent to determine the presence of recombinant yeast host cells in the specimen; (b) culturing a second sample of the specimen in a permissive medium lacking the pro-cytotoxic agent to determine the total microbial population of the specimen; and (c) determining the presence of contaminating microorganisms in the specimen based on the determination made in steps (a) and (b). In an embodiment, the noxious gene is FCY1 and in yet a further embodiment, the pro- cytotoxic agent is 5-fluorocytosine (5-FC). In an embodiment, the noxious gene is FUR1 and in yet a further embodiment, the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5- FU). In an embodiment, the noxious gene comprises FUR1 and in yet a further embodiment, the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU).

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Figure 1 provides an embodiment of the process for monitoring and limiting microbial contamination during fermentation.

Figures 2A to 2C show culture results of contaminating yeasts (including strain PE-2) and strain M 10682 on medium lacking and comprising 5-fluorocytosine (5FC). (Figure 2A) Plates shown in the upper panel lack 5-FC and plates shown in the lowest panel include 500 mg/mL 5- FC. Various combinations of strains M 10682 and PE-2 ((i) 12% M10682, 88% PE-2; (ii) 26% M10682, 74% PE-2; (iii) 51 % M10682, 49% PE-2; (iv) 73% M 10682, 27% PE-2 and (v) 92% M10682, 8% PE-2) were made and plated. The number of colony-forming units is included for each plate. (Figure 2B) The plates shown in the upper panel lack 5-FC and allow the growth of all the yeast strains tested. The plates plate shown in the lowest panel include 5-FC and did not allow the growth of wild-type (e.g., non-recombinant) yeast host cell. (Figure 2C) Liquid medium lacking (i) or comprising 500 mg/mL 5-FC (ii) were either not inoculated (media alone) or inoculated with strain PE-2 or M 10682.

Figures 3A to 3C show the effect of 5-FC addition on mixed cultures of M 10682 and PE-2. M10682 was mixed with 1 % (Figure 3A), 5% (Figure 3B) or 10% (Figure 3C) PE-2 yeast and multiple rounds of fermentation and cell recycle were performed in an industrially sourced sugarcane supplemented with 0 (¨), 1 (□) or 4

ppm 5-FC. After each of four cycles of fermentation, the yeast were subcultured and grown without selection for seven generations. The relative strain abundance was then determined by quantitative PCR (qPCR). Results are shows as the percentage of the contaminant (PE-2) yeast detected in function of the amount of 5-FC used and the fermentation cycle.

Figure 4 shows the effect of a fur1 deletion on the resistance to 5-FU and ethanol production. Results are shown as the ethanol content (g/L) in function of the different strains or combinations of strains used. Dark grey bars provide the ethanol production of each strain/mixed population in the absence of 5-FU. Light gray bars provide the ethanol production of each strain/mixed population in the presence of 50 ppm of 5-FU.

Figures 5A and 5B show the effect of a furl deletion on the resistance to 5-FC. (Figure 5A) Strain PE-2 was mixed with strains M10682 or M15980 (at the ratio indicated on the figures) and fermentations were carried out at 33°C on an industrial sugarcane must or the same must supplemented with 0 or 10 ppm 5-FC. The final ethanol titers as measured by HPLC. Results are shown as the ethanol content (g/L) in function of the different strains or combinations of strains used in the absence (dark gray bar) or presence (light gray bars) of 5-FC. (Figure SB) The percentage of recombinant yeasts in the fermented yeast population before (dark gray bars) and following (light gray bars) treatment with 10 ppm 5FC as determined by selective agar plating. Results are shown as the percentage of the recombinant yeast in the yeast population in function of the combination of yeasts tested.

Figure 6 provide a diagram of the conversion of cytosine (left panel) or 5-fluorocytosine (5FC) / 5-fluorouracil (5FU) (right panel) during protein and DNA synthesis. As shown on the left panel, 5FU is being converted by the FUR1 protein into compounds (FUMP, FUDP and FdUMP) inhibit RNA or DNA synthesis are thus considered cytotoxic to a yeast cell expressing the FUR1 protein

DETAILED DESCRIPTION

Unless defined otherwise, 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 belongs. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

The terms“gene(s)” or“polynucleotide” or“polynucleotide sequence(s)” are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In the present disclosure, the gene is endogenous to the host cell and is thus located in the genome of the host cell.

In the context of the present disclosure, a“gene ortholog” is understood to be a gene in a different species that evolved from a common ancestral gene by speciation. It is understood that the protein encoded by a gene ortholog retains the same function as the protein encoded by the common ancestral gene.

In the context of the present disclosure, the yeast host cell is a recombinant microorganism, e.g., a yeast host cell in which a genetic modification has been introduced. When the genetic modification is aimed at reducing or inhibiting the expression of a specific targeted gene (which is endogenous to the host cell, such as, for example, a potentially noxious gene), the genetic modifications can be made in one or both copies of the targeted gene(s). Alternatively or in combination, the genetic mod ifi cation (s) can be made at one or both copies of a gene whose expression product modulates the expression or function of the target gene(s) (such as, for example, a gene encoding for an inhibitor of expression the target gene(s) or for an inhibitor for a protein encoded by the target gene(s)). Such genetic modification can result in the disruption of the open reading frame either by deleting one or more nucleic acid residues in the gene or adding one or more nucleic acid residues in the gene. When the genetic modification is aimed at increasing the expression of a specific targeted gene (which is considered heterologous to the host cell, such as, for example, a gene involved in the production of a biofuel), the genetic modification can be made in one or multiple genetic locations. In the context of the present disclosure, when recombinant yeast cell is qualified as being “genetically engineered”, it is understood to mean that it has been manipulated to either add at least one or more heterologous or exogenous nucleic acid residue (e.g., genetic addition) and/or removed at least one endogenous (or native) nucleic acid residue (e.g., genetic deletion). In some embodiments, the one or more nucleic acid residues that are added can be derived from an heterologous cell or the recombinant host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at a genomic location which is different than the native genomic location. In some embodiments, a single genetic modification can be made to increase the expression of a specific gene and decrease the expression of another specific gene. This can be done, for example, by introducing the coding sequence of the specific gene inside the coding sequence of the other specific gene which will ultimately cause the disruption of the reading frame of the other specific gene. The genetic modifications described herein did not occur in nature and are the results of in vitro manipulations of the yeast and/or of an adaptive evolution of a parental strain cultured in the presence of an evolution pressure (such as, for example, in the presence of one or more (pro-)cytotoxic compounds).

The recombinant yeast host cell can be obtained from any yeast host cell. In an embodiment, the recombinant yeast host cell can be used in the production of biofuels. Suitable yeast host cells can be, for example, from the genus Saccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Torula or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae, S. bulderi, S. bametti, S. exiguus, S. uvarum, S. diastaticus, C. utilis, K. lactis, K. marxianus or K. fragilis. In some embodiments, the yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In some embodiment, the yeast host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiment, the host cell can be an oleaginous microalgae host cell (e.g., for example, from the genus Thraustochytrium or Schizochytrium). In an embodiment, the recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae.

Colony-forming units. As used in the context of the present disclosure, colony forming units (or CFU) is a unit used to estimate the number of viable microorganism in a sample.

Cytotoxic/pro-cytotoxic agent. As used in the context of the present disclosure, a “cytotoxic agent” refers to any agent, usually a chemical compound, capable of reducing the growth and/or the viability of a contaminating microorganisms. The cytotoxic agent is generated by the metabolism of a “pro-cytotoxic agent” by the enzymatic activity of a protein encoded by a noxious gene. The gene is considered being “noxious” to the contaminating microorganism express it as it allows the metabolism of the pro-cytotoxic agent into the cytotoxic agent in the contaminating microorganism. The cytotoxic agent does not reduce the growth and/or the viability of the recombinant yeast host cell because the recombinant yeast host cell includes a genetic modification so as to allow the reduction or the inhibition of expression of the noxious gene. The reduction or inhibition of the expression of the noxious gene renders the recombinant yeast host cell resistant (at least in part) to the cytotoxic agent because the recombinant yeast host cell lack the ability to metabolically convert the pro-cytotoxic agent into the cytotoxic agent. It will be understood by those skilled in the art that, in the methods and processes of the present disclosure, the agent used must be carefully selected to match the noxious gene being inhibited in the recombination yeast host cell and vice versa. In an embodiment, the noxious gene is a noxious gene encoding a key enzyme for the production of a monomer used in biosynthesis

(such as, for example, amino acids and nucleotides). In Saccharomyces cerevisiae, noxious genes include, but are not limited to FCY1 (which can be used, in some embodiments, in combination with the pro-cytotoxic agent 5-FC), FUR1 (which can be used, in some embodiments, in combination with the pro-cytotoxic agent 5-FC and/or 5-FU), UR A3 (which can be used, in some embodiments, in combination with the pro-cytotoxic agent 5-fluoroorotic acid or 5-FOA), LYS2, LEU2, TRP1 , HIS3, MET15 and ADE2. In the context of the present disclosure, it is contemplated that the expression of more than one noxious gene be inactivated or reduced. For example, in some embodiments of the present disclosure, it is contemplated that both FCY1 and FUR1 (which are considered noxious genes) be inactivated in the recombinant yeast host cell.

Microbial population. As used in the context of the present disclosure, the term “microbial population” refers to a number microorganisms (irrespective of the fact that some members of the population may be recombinant yeast host cells or contaminating microorganisms). The expression“total microbial population” refers to the total number of microorganism in a specific volume or weight, usually a sample of the cultured or fermented medium (also referred to as a fermentation medium).

Microbial contamination. As used in the present disclosure, the term“microbial contamination” refers to a subpopulation of the microbial population distinct from the recombinant yeast host cells described herein. The microbial contamination is composed of contaminating microorganisms which can be yeasts and lack the genetic modification present in the recombinant yeast host cells rendering them resistant to the cytotoxic agent. Contaminating microorganisms include contaminating yeasts (such as, for example, from the Saccharomyces sp. (e.g., Saccharomyces cerevisiae ), the Dekkera sp. (e.g., Dekkera bruxellensis ), the Candida sp. (e.g., Candida krusei) and/or the Schizosaccharomyces sp. (e.g., Schizosaccharomyces pombe ) as well as bacteria which express a protein with similar function to the noxious protein that has been inactivated in the recombinant yeast host cell. In an embodiment, the contaminating microorganisms are yeasts. In a specific embodiment, the microbial contamination comprise a majority of yeasts from the Saccharomyces sp. and a minority of yeasts from the non -Saccharomyces sp. (such as, for example, from the Dekkera sp., the Candida sp. and/or the Schizosaccharomyces sp.). In yet another embodiment, the microbial contamination comprise at least 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95% or more of yeasts from the Saccharomyces sp.. In still another embodiment, the microbial contamination comprises no more than 40%, 30%, 20%, 10%, 5% or less of yeasts from the non- Saccharomyces sp.

Method for determining/monitoring microbial contamination

The present disclosure includes a method for determining the presence and/or monitoring a microbial contamination from a cultured medium or a sample thereof. The method is based on the use of a recombinant yeast host cell bearing a specific genetic modification for decreasing or impeding the expression of one or more noxious gene and ultimately conferring resistance to a cytotoxic agent in the recombinant yeast host cell.

In the context of the present disclosure, and in cells lacking the one or more genetic modification, a pro-cytotoxic agent is metabolized by the polypeptide encoded by the noxious gene to generate the cytotoxic agent. As such, limiting the expression of the noxious gene in the recombinant yeast host cell inhibits or impedes the expression/activity of the polypeptide encoded by the noxious gene which alters or reduces the conversion of a pro-cytotoxic agent into a cytotoxic agent which ultimately confers an (at least partial) resistance to the cytotoxic agent.

Broadly, the determination method comprises culturing the microbial population obtained from a specimen, a cultured medium, a fermented medium or a sample thereof in a selective medium (comprising the pro-cytotoxic agent for allowing the growth of the recombinant yeast host cell comprising the genetic modification as described herein and inhibiting the growth of contaminating microorganisms which do not possess the genetic modification) and/or in a permissive medium (lacking the pro-cytotoxic agent for allowing the growth of microorganisms present in the cultured medium or the sample thereof, irrespective of the fact that they bear or not the genetic modification). This dual culture system allows discriminating the microbial population between the recombinant yeast host cells (capable of growing in both media) and contaminating microorganisms (capable of growing in the permissive medium only). The method also comprises determining the proportion of recombinant yeast host cells and/or contaminating microorganisms in the microbial population of the specimen, the cultured medium or the fermented medium. Such determination can be made by methods known in the art such as, for example, assessing the number of colony forming units on cultured plates, the optical density in liquid medium, the number of cells using an hemocytometer, nucleic acid detection (amplification, sequencing, micro-array, etc.), etc. Such determination can be presented, for example, as the ratio of the number of cells/CFU from the recombinant yeasts to the total number of cells/CFU of the medium or the sample, the ratio of the number of cells/CFU from the contaminating microorganisms to the total number of cells/CFU of the medium or the sample, the ratio of the number of cells/CFU from the recombinant yeasts to the number of cells/CFU from the contaminating microorganisms and/or the ratio of the number of cells/CFU from the microorganisms to the number of cells/CFU from the recombinant yeasts. In another example, the determination can be presented as the percentage of the number of cells/CFU of the contaminating microorganisms with respect to the total number of cells/CFU of the medium or the sample, the percentage of the number of cells/CFU of the recombinant yeast host with respect to the total number of cells/CFU of the medium or the sample. In still another embodiment, the determination is presented as the percentage of the number of cells of the contaminating microorganisms with respect to the total number of cells of the medium or the sample. The determination can be repeated in time and in such instances, it is referring to as “monitoring” the culture.

The method can be performed to determine the presence and extent of a microbial contamination by contaminating microorganisms and, as indicated above, can be used to monitor the evolution of the microbial contamination in time. The method is especially useful for monitoring continuous cultures of the recombinant yeast host cell. As used in the context of the present disclosure, a“continuous culture” of the recombinant yeast host cell refers to a culture lasting at least a fermentation cycle and is subject to take-over from wild-type contaminating microorganisms, such as, for example, by wild-type yeasts. Continuous cultures can include fed batch cultures in which the yeast cells are recycled to inoculate a new fed batch. Continuous cultures are performed, for example, in the Brazilian biofuel processes, corn fermentation as well as in other long running ethanol process.

Recombinant yeast host cell

As described herein, he recombinant yeast host cell bears one ore more genetic modification for decreasing or impeding the expression of one or more endogenous noxious gene. Suitable endogenous noxious genes include, but are not limited to those involved in de novo nucleic acid synthesis such as uridine synthesis (FCY1 , FUR1) or adenine synthesis as well as those involved in amino acid synthesis (URA3, LYS2, LEU2, TRP1 , HIS3, MET15 and ADE2). The genetic modification can be made in one or more of the noxious genes as well as in one or more copies of the noxious genes. In an embodiment, the genetic modification is made to all copies of the selected noxious gene(s). In an embodiment, the genetic modification is a genetic addition within the open reading frame of the noxious gene so as to disrupt the reading frame of such gene.

In an embodiment, the recombinant yeast host cell can bear a genetic modification for decreasing/impeding the expression of a gene encoding a cytosine deaminase, such as, for example, the FCY1 gene or a gene ortholog of the FCY1 gene. In Saccharomyces cerevisiae, the FCY1 gene encodes a cytosine deaminase capable of metabolizing the pro-cytotoxic agents, 5-fluorocytosine and 5-f!uorocytidine, into cytotoxic agents, 5-fluorouracil and 5- fluorouridine, respectively. The genetic modification can be made in one or more of the following genes Saccharomyces cerevisiae Gene ID 856175, Scheffersomyces stipitis Gene ID 4839675 and 4838793, Sugiyamaella lignohabitans Gene ID: 30036201 , Saccharomyces eubayanus Gene ID: 28934651 , Beauveria bassiana Gene ID: 19888208, Purpureocillium lilacinumor Gene ID: 28882931 , Ostreococcus tauri Gene ID: 9837926 and 9833302 as well as their corresponding gene orthologs. Alternatively or in combination, the genetic modification can be made in one or more genes encoding the following proteins identified by their GenBank Accession Number: NP_015387.1 , AJW23097.1 , AJV96709.1 , EGA60101.1 , ATB23854.1 , EHN04073.1 , AAG33626.1 , EGA72733.1 , SCN22162.1 , AFM78648.1 , XP_018219080.1 , EHM99818.1 , XP_003680593.1 , XP_003683874.1 , XP_003667919.1 , SMN18141.1 ,

XP_003674048.1 , XP_001644868.1 , XP_445483.1 , XP_003958642.1 , SCW01268.1 , ABG43006.1 , CUS23860.1 , XP_002554144.1 , SCV05713.1 , SCU92348.1 , XP_002496341.1 ,

SCU81687.1 , AQZ14233.1 , GAV53400.1 , CDH13752.1 , XP_022630113.1 , SCU82976.1 , AQZ10489.1 , XP_022465730.1 , XP_451904.1 , CDO92427.1 , XP_022678383.1 , SCV03733.1 , AG012921.1 , CR_004177972.1 , CR_019038655.1 , NR_983768.2, CR_011272138.1 ,

CCE84718.1 , CR_001385070.2, CR_020078665.1 , CR_020071353.1 , CR_007377322.1 ,

CR_018209416.1 , CR_013936499.1 , CR_015469400.1 , ABD24095.1 , OVF09408.1 ,

OEJ85321 .1 , KDQ23147.1 , ODV98554.1 , CR_018711764.1 , EDK39700.2, CR_001484417.1 , SGZ47344.1 , CR_002770827.1 , CR_006687734.1 , ONH68939.1 , CR_016213901.1 ,

PIS48695.1 , CR_018987201.1 , GAT44865.1 , CR_016229010.1 , ORY62206.1 ,

CR_016261374.1 , OCW31955.1 , CDR37774.1 , GAD94373.1 , CR_009546940.1 ,

CR_018191494.1 , KUL86194.1 , KKU36622.1 , KZR31907.1 , EMR81126.1 , KIW62513.1 , GAM38076.1 , KUI66787.1 , PMD17950.1 , EMG45712.1 , CR_022501696.1 , CR_013319060.1 , CR_002150180.1 , CR_002484082.1 , CR_020066952.1 , KKU16093.1 , OAL07706.1 ,

CAE82258.1 , EKG21993.1 or PIL30025.1 . The genetic modification can be made in the FCY1 gene (or the FCY1 gene ortholog) as well as in one or more copies of the FCY1 gene (or the FCY1 gene ortholog). In an embodiment, the genetic modification is made to all copies of the FCY1 gene (or the FCY1 gene ortholog). In an embodiment, the genetic modification is a genetic addition within the open reading frame of the FCY1 gene (or the FCY1 gene ortholog) so as to disrupt the reading frame of the FCY1 gene (or the FCY1 gene ortholog) thereby inhibiting its expression. In combination with a pro-cytotoxic agent, recombinant yeast host cell having a genetic modification in the FCY1 gene (or a FCY1 gene ortholog) are especially useful for limiting microbial contamination in continuous cultures having less than 10% microbial contamination.

In an embodiment, the recombinant yeast host cell can bear a genetic modification for decreasing the expression of an uracil phosphoribosyltransferase, such as the FUR1 gene or a gene ortholog of the FUR1 gene. In Saccharomyces cerevisiae, the FUR1 gene encodes an uracil phosphoribosyltransferase capable of metabolizing the pro-cytotoxic agents, 5-fluorouracil into 5-fluorouridine monophosphate and 5-fluorodeoxyuridine monophosphate. The genetic modification can be made in one or more of the following genes Saccharomyces cerevisiae Gene ID: 856529, Candida albicans Gene ID: 3646348, Scheffersomyces stipitis Gene ID: 4839411 , Sugiyamaella lignohabitans Gene ID: 30037338, Saccharomyces eubayanus Gene ID: 28931669, Candida auris Gene ID: 28873876, Candida orthopsilosis Gene ID: 14538621 , Zymoseptoria tritici Gene ID: 13394585 as well as their corresponding orthologs. Alternatively or in combination, the genetic modification can be made in one or more genes encoding the following proteins identified by their GenBank Accession Number: NP_011996.2, AAA34611 .1 , EGA78586.1 , EJS43479.1 , AAG33626.1 , AAB19947.2, EJT44383.1 , ATB23854.1 ,

SCN22162.1 , AFM78648.1 , XP_018221908.1 , EGA74672.1 , XP_001646180.1 , XP_003675167.1 , XP_003686112.1 , A5H0J4.1 , XP_447193.1 , XP_003668723.1 ,

XP_022466023.1 , SMN18920.1 , XP_003957792.1 , XP_022628074.1 , SCU87814.1 ,

SCU85479.1 , XP_002498244.1 , GAV55604.1 , CDF91960.1 , GAV50813.1 , SJM86598.1 , XP_003670868.1 , SCU95973.1 , SCV02910.1 , XP_002552045.1 , SCW03519.1 , XP_003647576.1 , XP_017986375.1 , CUS22339.1 , XP_003679822.1 , XP_022674246.1 ,

SCU80196.1 , XP_454985.1 , CD095124.1 , AGO13201 .1 , NP_985599.1 , XP_019041613.1 , XP_020049305.1 , ABD19514.1 , XP_002615165.1 , XP_011274553.1 , XP_018985203.1 ,

ANZ74534.1 , XP_015465817.1 , CCE84646.1 , OEJ88599.1 , EHN02058.1 , SGZ51144.1 , XP_002489914.1 , ODV98452.1 , XP_001385142.1 , CCE43978.1 , XP_506088.1 ,

XP_003867375.1 , XP_018710073.1 , ODQ68545.1 , XP_018172396.2, XP_020073271.1 , XP_002770455.1 , XP_002420612.1 , EMG46465.1 , XP_006685579.1 , OUM53290.1 ,

XP_712023.1 , XP_002548392.1 , GNH70060.1 , XP_007375441 .1 , XP_020064281 .1 ,

XP_001524806.1 , CDR37616.1 , OWB76646.1 , XP_001482408.1 , OWB55638.1 , KGR11 196.1 , XP_020074370.1 , XP_022457337.1 , CDG55385.1 , XP_013932538.1 , KKA25964.1 ,

CZR59070.1 , PMD64816.1 , CCD51448.1 , XP_007838875.1 , XP_018074385.1 ,

XP_001598104.1 , PHH51712.1 , CEJ94560.1 , ORY06946.1 , EIF48879.1 , OIW29874.1 or XP_008079195.1. The genetic modification can be made in the FUR1 gene (or the FUR1 gene ortholog) as well as in one or more copies of the FUR1 gene (or the FUR1 gene ortholog). In an embodiment, the genetic modification is made to all copies of the FUR1 gene (or the FUR1 gene ortholog). In an embodiment, the genetic modification is a genetic addition within the open reading frame of the FUR1 gene (or the FUR1 gene ortholog) so as to disrupt the reading frame of the FUR1 gene (or the FUR1 gene ortholog) thereby inhibiting its expression. In combination with a pro-cytotoxic agent, recombinant yeast host cell having a genetic modification in the FUR1 gene (or a FUR1 gene ortholog) are especially useful for limiting microbial contamination in continuous cultures even though having more than 10% microbial contamination.

In still another embodiment, the recombinant yeast can bear at least two distinct genetic modifications: a first one for decreasing or impeding the expression of a FCY1 gene (or a corresponding gene ortholog) and a second one for decreasing or impeding the expression of a FCU1 gene (or a corresponding gene ortholog). In this embodiment, the genetic modifications are made to both the FCY1 and the FUR1 genes (or their corresponding gene orthologs) as well as in one or more copies of the FCY1 and the FUR1 genes (or their corresponding gene orthologs). In an embodiment, the genetic modification is made to all copies of the FCY1 and the FUR1 genes (or their corresponding gene orthologs). In an embodiment, the genetic modification is a genetic addition within the open reading frame of the FCY1 and the FUR1 genes (or their corresponding gene orthologs) so as to disrupt the reading frame of both the FCY1 and the FUR1 gene (or their corresponding gene orthologs). In combination with a pro- cytotoxic agent, recombinant yeast host cell having a genetic modification in both the FCY1 and the FUR1 gene (or their corresponding gene orthologs) are especially useful for limiting microbial contamination in continuous cultures even though having more than 10% microbial contamination. The recombinant yeast host cell of the present disclosure can also include additional genetic modifications, for example, a genetic modification for increasing the activity of a protein which functions to import glycerol and/or a genetic modification for decreasing its NAD-dependent glycerol-3-phosphate activity in high osmotic conditions, as indicated in PCT patent application published under WO2018/215956 and filed on May 23, 2018.

The recombinant microbial host cell can have at least one genetic modification allowing it to increase the (biological) activity of a protein which functions to import glycerol (e.g., actively transport glycerol inside the cell) and/or decrease the (biological) activity of a protein which functions to export glycerol (e.g., actively transport glycerol inside the cell). Still in the context of the present disclosure, the activity of the protein functioning to import/export glycerol in the recombinant microbial host cell is modulated in glycolytic conditions.

In an embodiment, the recombinant microbial host cells has at least one genetic modification allowing it to increase the (biological) activity of a protein which functions to import glycerol (e.g., actively transport glycerol inside the cell). Still in the context of the present disclosure, the activity of the protein functioning to import glycerol in the recombinant microbial host cell is increased in glycolytic conditions. The STL1 protein is an exemplary protein which functions to import glycerol.

In another embodiment, the recombinant microbial host cells has at least one genetic modification allowing it to decrease the (biological) activity of a protein which functions to export glycerol (e.g., actively transport glycerol outside the cell). Still in the context of the present disclosure, the activity of the protein functioning to export glycerol in the recombinant microbial host cell is decreased in glycolytic conditions. The FPS1 protein is an exemplary protein which functions to export glycerol. The FPS1 protein a channel protein located in the plasma membrane that controls the accumulation and release of glycerol in yeast osmoregulation. Null mutants of this strain accumulate large amounts of intracellular glycerol, grow much slower than wild-type, and consume the sugar substrate at a slower rate. As such, the genetic modification can include reducing or deleting the expression of the gene encoding the FPS1 protein during glycolytic conditions.

As used in the context of the present disclosure, the expression“glycolytic conditions” refers to the presence of sufficient glucose in the environment surrounding the recombinant microbial host cell to trigger the uptake of that glucose by the cell. The increase in glycerol importing activity can be observed with respect to the same recombinant microbial cell that is not undergoing glycolysis (for example during the propagation phase of the recombinant microbial cell or in the absence of glucose). This increase can also be observed with respect to a corresponding recombinant microbial host cell lacking the genetic modification. In the context of the present disclosure, it is not necessary that the increase in activity of the protein functioning to import glycerol be limited to circumstances in which the recombinant microbial host cell be in glycolytic conditions but it is important that the increase in activity be observed when the recombinant microbial host cell is placed in glycolytic conditions.

The recombinant microbial host cells of the present disclosure can include a genetic modification to introduce (one or more copies of) of an heterologous nucleic acid molecule encoding an heterologous protein functioning to import glycerol and/or to replace the promoter of the gene encoding the native protein functioning to import glycerol with a glycolytic promoter.

In order to increase the activity of the protein functioning to import glycerol, it is possible to include, in the recombinant microbial host cell, one or more copies of an heterologous nucleic acid molecule encoding the protein functioning to import glycerol. For example, the recombinant microbial host cell can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In an embodiment, the recombinant microbial host cell comprises between four and eight copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In an embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) two copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In another embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) three copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In yet another embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) four copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In still another embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) five copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In a further embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) six copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In yet a further embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) seven copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. In still a further embodiment, the recombinant microbial host cell comprises at least (and in some additional embodiments no more than) eight copies of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol. The heterologous nucleic acid molecule can be independently replicating or integrated in the recombinant microbial host cell. When the heterologous nucleic acid molecule is integrated in the recombinant microbial host cell, it is preferably positioned at neutral integration site. When more than one copy of the heterologous nucleic acid molecule encoding the protein functioning to import glycerol is introduced in the recombinant microbial host cell, each of the copy can be integrated at one or more (the same or different) integration sites.

In order to achieve the expression (or, in some embodiments, the overexpression) of the activity of the protein functioning to import glycerol in glycolytic conditions, it may be necessary to include a glycolytic promoter to control the expression of the gene encoding the protein functioning to import glycerol. In the context of the present disclosure, a“glycolytic promoter” is a promoter (or a combination of promoters) allowing the expression (or, in some embodiments, the overexpression) of a gene when the recombinant microbial cell is in placed in glycolytic conditions. The glycolytic promoter can be included in the recombinant microbial host cell either to control the expression of a native and/or an heterologous gene encoding the protein functioning to import glycerol. The glycolytic promoter can be a constitutive promoter or a glucose-inducible promoter. Glycolytic promoters exclude glucose-repressible promoters. Glucose-inducible promoters are usually associated with genes encoding enzymes in the glycolytic pathway and promoters controlling the expression of enzymes which are upregulated in the glycolytic pathway can be used in the recombinant microbial host cell of the present disclosure. Enzymes of the glycolytic pathway whose expression is upregulated in the presence of glucose include, but are not limited to, those encoded by an alcohol dehydrogenase gene, a glucose-6-phosphate isomerase gene, a phosphofructokinase gene, an aldolase gene, a triosephosphate isomerase gene, a glyceraldehyde-3-phosphate dehydrogenase gene, a 3- phosphoglycerate kinase gene, a phosphoglycerate mutase, an enolase and a pyruvate kinase gene. As such, in the context of the present disclosure, the glycolytic promoter can be a promoter (or a combination of promoters) from an alcohol dehydrogenase gene, a glucose-e- phosphate isomerase gene, a phosphofructokinase gene, an aldolase gene, a triosephosphate isomerase gene, a glyceraldehyde-3-phosphate dehydrogenase gene, a 3-phosphoglycerate kinase gene, a phosphoglycerate mutase, an enolase and/or a pyruvate kinase gene.

In Saccharomyces cerevisiae, enzymes of the glycolytic pathway whose expression is upregulated in the presence of glucose include, but are not limited to, those encoded by a ADH1 gene, a PGI1 gene, a PFK1 gene, a PFK2 gene, a FBA1 gene, a TPI1 gene, a TDH1 gene, a TDH2 gene, a TDH3 gene, a PGK1 gene, a GPM1 gene, a ENG1 gene, a ENG2 gene, a PYK2 gene and a CDC19 gene. As such, in the context of the present disclosure, the glycolytic promoter can be a promoter (or a combination of promoters) from a ADH1 gene (referred to as the ADH1 promoter or adhl p), a PGI1 gene (referred to as the PGI1 promoter or pgi1p), a PFK1 gene (referred to as the PFK1 promoter or pfki1p), a PFK2 gene (referred to as the PFK2 promoter or the pfk2p), a FBA1 gene (referred to as the FBA1 promoter or fbal p), a TPI1 gene (referred to as a TPI1 promoter or tpi1 p), a TDH1 gene (referred to as the TDH1 promoter or tdhl p), a TDH2 gene (referred to as the TDH2 promoter or tdh2p), a TDH3 gene (referred to as the TDH3 promoter or tdh3p), a PGK1 gene (referred to as the PGK1 promoter or pgkl p), a GPM1 gene (referred to as the GPM1 promoter or gpml p), a EN01 gene (referred to as the EN01 promoter or eno1 p), a ENG2 gene (referred to as the ENG2 promoter or eno2p), a PYK2 gene (referred to as the PYK2 promoter or pyk2p) and/or a CDC19 gene (referred to as the CDC19 or odc19p).

Exemplary proteins capable of functioning to import glycerol include aquaporins as well as glycerol facilitators. The FPS1 protein (encoded by Gene ID 850683 in Saccharomyces cerevisiae ) is a glycerol facilitator capable of importing glycerol. As such, the protein capable of functioning to import glycerol can be a FPS1 protein or a protein encoded by a FPS1 gene ortholog. The FPS1 protein can be derived, for example, from Saccharomyces cerevisiae or a corresponding ortholog found in Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and/or Cyberlindnera jadinii

Another exemplary protein capable of functioning to import glycerol is the glucose-inactivated glycerol/proton symporter STL1. The native function of the STL1 protein is the uptake of glycerol from the extracellular environment. STL1 is a member of the Sugar Porter Family which is part of the Major Facilitator Superfamily (MFS). STL1 transports glycerol by proton symport meaning that the glycerol and protons are cotransported through STL1 into the cell. In S. cerevisiae, STL1 expression and glycerol uptake is typically repressed when there are other carbon sources such as glucose available. When the cells undergo high osmotic shock STL1 is expressed in order to help deal with the osmotic shock by transporting the osmoprotectant glycerol into the cell and increasing the intracellular glycerol concentration. In the context of the present disclosure, the protein functioning to import glycerol can be the STL1 protein, a variant of the STL1 protein or a fragment of the STL1 protein. In the embodiments in which the protein functioning to import glycerol is a variant or a fragment of the STL1 protein, the variant or the fragment need to exhibit at least some of the biological activity of the native STL1 protein, namely the ability to act as a proton symport as indicated above.

The heterologous protein functioning to import glycerol can be encoded by a STL1 gene. The STL1 protein is natively expressed in yeasts and fungi, therefore the heterologous protein functioning to import glycerol can be derived from yeasts and fungi. STL1 genes encoding the STL1 protein include, but are not limited to, Saccharomyces cerevisiae Gene ID: 852149, Candida albicans, Kluyveromyces lactis Gene ID: 2896463, Ashbya gossypii Gene ID: 4620396, Eremothecium sinecaudum Gene ID: 28724161 , Torulaspora delbrueckii Gene ID: 11505245, Lachancea thermotolerans Gene ID: 8290820, Phialophora attae Gene ID: 28742143, Penicillium digitatum Gene ID: 26229435, Aspergillus oryzae Gene ID: 5997623, Aspergillus fumigatus Gene ID: 3504696, Talaromyces atroroseus Gene ID: 31007540, Rasamsonia emersonii Gene ID: 25315795, Aspergillus flavus Gene ID: 7910112, Aspergillus terreus Gene ID: 4322759, Penicillium chrysogenum Gene ID: 8310605, Altemaria alternata Gene ID : 29120952, Paraphaeosphaeria sporulosa Gene ID: 28767590, Pyrenophora tritici- repentis Gene ID: 6350281 , Metarhizium robertsii Gene ID: 19259252, Isaria fumosorosea Gene ID: 30023973, Cordyceps militaris Gene ID: 18171218, Pochonia chlamydosporia Gene ID : 28856912, Metarhizium majus Gene ID: 26274087, Neofusicoccum parvum Gene 10:19029314, Diplodia corticola Gene ID: 31017281 , Verticillium dahliae Gene ID: 2071 1921 , Colletotrichum gloeosporioides Gene ID: 18740172, Verticillium albo-atrum Gene ID: 9537052, Paracoccidioides lutzii Gene ID: 9094964, Trichophyton rubrum Gene ID: 10373998, Nannizzia gypsea Gene ID: 10032882, Trichophyton verrucosum Gene ID: 9577427, Arthroderma benhamiae Gene ID: 9523991 , Magnaporthe oryzae Gene ID: 2678012, Gaeumannomyces graminis var. tritici Gene ID: 20349750, Togninia minima Gene ID: 19329524, Eutypa lata Gene ID: 19232829, Scedosporium apiospermum Gene ID: 27721841 , Aureobasidium namibiae Gene ID: 25414329, Sphaerulina musiva Gene ID: 27905328 as well as Pachysolen tannophilus GenBank Accession Numbers JQ481633 and JQ481634, Saccharomyces paradoxus STL1 and Pichia sorbitophilia. In an embodiment, the STL1 protein is encoded by Saccharomyces cerevisiae Gene ID: 852149.

The heterologous protein functioning to import glycerol can be encoded by a STL1 gene as indicated herein or a STL1 gene ortholog. The heterologous protein functioning to import glycerol can be a STL1 protein as defined herein, a variant of the STL1 protein and/or a fragment of the STL1 protein. In addition, when more than one copy of the heterologous STL1 is included in the recombinant microbial cell, the plurality of heterologous nucleic acid molecules encoding the STL1 protein could be the same or different, integrated at the same or different integration sites.

The recombinant microbial host cell can also have a further modification allowing it to decrease its NAD-dependent glycerol-3-phosphate (biological) activity in high osmotic conditions. Importantly, the recombinant microbial host cell may retain substantially the same NAD- dependent glycerol-3-phosphate (biological) activity in normal to low osmotic conditions. In yet a further embodiment, the recombinant microbial host cell of the present disclosure can express at least one GPD protein (which can be native or heterologous to the microbial host cell).

As used herein, the expression “high osmotic conditions” refers to the presence of a high osmotic pressure, usually caused by an increase in the solute concentration in the environment surrounding the recombinant microbial host cell. In some embodiments, “high osmotic conditions” are associated with an upregulation of the HOG, a concentration of sugars higher than about 50 g/L and/or equivalent to at least 1 g/L of salt (such as NaCI) when the recombinant microbial host cell is a yeast host cell. This decrease in NAD-dependent glycerol-3- phosphate activity can be observed with respect to the same recombinant cell in normal or low osmotic conditions or with respect to a recombinant microbial host cell lacking the genetic modification. As also used in the present disclosure, the expression “normal or low osmotic conditions” refers to conditions that are not associated with high osmotic pressure. Most mammalian cells express two different glycerol-3-phosphate dehydrogenases (GPDs) which are necessary for glycerol production and they are expressed in response to different cellular signals: the GPD1 and the GPD2 proteins. Both proteins share 75% amino acid identity and, while they catalyze the same reaction, the differences in their amino acid sequence make them more efficient enzymes under the environmental conditions that induce their expression. GPD2 is known to be unable to fully substitute for GPD1 in the production of osmotically induced glycerol production suggesting that this enzyme has lower activity than GPD1 under osmotic stress.

The recombinant microbial host cells of the present disclosure can include a genetic modification to inhibit (at least partially or totally) the expression of the NAD-dependent glycerol- 3-phosphate activity 1 (GPD1) protein or a GPD1 gene ortholog. The genetic modification can include the deletion, deletion or substitution of one or more of a nucleic acid residue(s) in a gene (or a gene orholog) encoding the GPD1 protein (particularly in the gene’s coding sequence) which would cause a reduction in the activity of the GPD1 protein in high osmotic conditions. In an embodiment, the genetic modification can include the deletion of all of the coding sequence of a gene (or a gene ortholog) encoding the GPD1 protein. Alternatively or in combination, the recombinant microbial host cell can express an heterologous GPD1 protein variant or fragment having a reduced activity during high osmotic conditions when compared to the native GPD1 protein.

The GPD1 protein is natively expressed in yeasts, fungi, mammalian and plant cells. GPD1 genes encoding the GPD1 protein include, but are not limited to Saccharomyces cerevisiae Gene ID: 851539, Schizosaccharomyces pombe Gene ID: 2540547, Schizosaccharomyces pombe Gene ID: 2540455, Neurospora crassa Gene ID: 3873099, Candida albicans Gene ID: 3643924, Scheffersomyces stipitis Gene ID: 4840320, Spathaspora passalidarum Gene ID: 18874668, Trichoderma reesei Gene ID: 18482691 , Nectria haematococca Gene ID: 9668637, Candida dubliniensis Gene ID: 8046432, Chlamydomonas reinhardtii Gene ID: 5716580, Brassica napus Gene ID: 106365675, Chlorella variabilis Gene ID: 17355036, Brassica napus Gene ID: 106352802, Mus musculus Gene ID: 14555, Homo sapiens Gene ID: 2819, Rattus norvegicus Gene ID: 60666, Sus scrofa Gene ID: 100153250, Gallus gallus Gene ID: 426881 , Bos taurus Gene ID: 525042, Xenopus tropicalis Gene ID: 448519, Pan troglodytes Gene ID: 741054, Canis lupus famiiiaris Gene ID: 607942, Callorhinchus milii Gene ID: 103188923, Columba livia Gene ID: 102088900, Macaca fascicularis Gene ID: 101865501 , Myotis brandtii Gene ID: 102257341 , Heterocephalus glaber Gene ID: 101702723, Nannospalax galili Gene ID: 103746543, Mustela putorius furo Gene ID: 101681348, Callithrix jacchus Gene ID: 100414900, Labrus bergylta Gene ID: 109980872, Monopterus albus Gene ID: 109969143, Castor canadensis Gene ID: 109695417, Paralichthys olivaceus Gene ID: 109635348, Bos indicus Gene ID: 109559120, Hippocampus comes Gene ID: 109507993, Rhinolophus sinicus Gene ID: 109443801 , Hipposideros armiger Gene ID: 109393253, Crocodylus porosus Gene ID: 109324424, Gavialis gangeticus Gene ID: 109293349, Panthera pardus Gene ID: 109249099, Cyprinus carpio Gene ID: 109094445, Scleropages formosus Gene ID: 108931403, Nanorana parkeri Gene ID: 108789981 , Rhinopithecus bieti Gene ID: 108543924, Lepidothrix coronata Gene ID: 108509436, Pygocentrus nattereri Gene ID: 108444060, Manis javanica Gene ID: 108406536, Cebus capucinus imitator Gene ID: 108316082, lctalurus punctatus Gene ID: 108255083, Kryptolebias marmoratus Gene ID: 108231479, Miniopterus natalensis Gene ID: 107528262, Rousettus aegyptiacus Gene ID: 107514265, Cotumix japonica Gene ID: 107325705, Protobothrops mucrosquamatus Gene ID: 107302714, Parus major Gene ID: 107215690, Marmota marmota marmota Gene ID: 107148619, Gekko japonicus Gene ID:

107122513, Cyprinodon variegatus Gene ID: 107101128, Acinonyx jubatus Gene ID: 106969233, Poecilia latipinna Gene ID: 106959529, Poecilia mexicana Gene ID: 106929022, Calidris pugnax Gene ID: 106891167, Sturnus vulgaris Gene ID: 106863139, Equus asinus Gene ID: 106845052, Thamnophis sirtalis Gene ID: 106545289, Apteryx australis mantelli Gene ID: 106499434, Anser cygnoides domesticus Gene ID: 106047703, Dipodomys ordii Gene ID: 105987539, Clupea harengus Gene ID: 105897935, Microcebus murinus Gene ID: 105869862, Propithecus coquereli Gene ID: 105818148, Aotus nancymaae Gene ID: 105709449, Cercocebus atys Gene ID: 105580359, Mandrillus leucophaeus Gene ID: 105527974, Colobus angolensis palliatus Gene ID: 105507602, Macaca nemestrina Gene ID: 105492851 , Aquila chrysaetos canadensis Gene ID: 105414064, Pteropus vampyrus Gene ID: 105297559, Camelus dromedanus Gene ID: 105097186, Camelus bactnanus Gene ID: 105076223, Esox lucius Gene ID: 105016698, Bison bison bison Gene ID: 105001494, Notothenia coriiceps Gene ID: 104967388, Larimichthys crocea Gene ID: 104928374, Fukomys damarensis Gene ID: 04861981 , Haliaeetus leucocephalus Gene ID: 104831 135, Corvus comix comix Gene ID: 104683744, Rhinopithecus roxellana Gene ID: 104679694, Balearica regulorum gibbericeps

Gene ID: 104630128, Tinamus guttatus Gene ID: 104575187, Mesitomis unicolor Gene ID: 104539793, Antrostomus carolinensis Gene ID: 104532747, Buceros rhinoceros silvestris Gene ID: 104501599, Chaetura pelagica Gene ID: 104385595, Leptosomus discolor Gene ID: 104353902, Opisthocomus hoazin Gene ID: 104326607, Charadrius vociferus Gene ID: 104284804, Struthio camelus australis Gene ID: 104144034, Egretta garzetta Gene ID:

104132778, Cuculus canorus Gene ID: 104055090, Nipponia nippon Gene ID: 104011969, Pygoscelis adeliae Gene ID: 103914601 , Aptenodytes forsteri Gene ID: 103894920, Serinus Canada Gene ID: 103823858, Manacus vitellinus Gene ID: 103760593, Ursus maritimus Gene ID: 103675473, Corvus brachyrhynchos Gene ID: 103613218, Galeopterus variegatus Gene ID: 103598969, Equus przewalskii Gene ID: 103546083, Calypte anna Gene ID: 103536440,

Poecilia reticulata Gene ID: 103464660, Cynoglossus semilaevis Gene ID: 103386748, Stegastes partitus Gene ID: 103355454, Eptesicus fuscus Gene ID: 103285288, Chiorocebus sabaeus Gene ID: 103238296, Orycteropus afer afer Gene ID: 103194426, Poecilia formosa Gene ID: 103134553, Erinaceus europaeus Gene ID: 103118279, Lipotes vexillifer Gene ID: 103087725, Python bivittatus Gene ID: 103049416, Astyanax mexicanus Gene ID: 103021315, Balaenoptera acutorostrata scammoni Gene ID: 103006680, Physeter catodon Gene ID: 102996836, Panthera tigris altaica Gene ID: 102961238, Chelonia mydas Gene ID: 102939076, Peromyscus maniculatus bairdii Gene ID: 102922332, Pteropus alecto Gene ID: 102880604, Elephantulus edwardii Gene ID: 102844587, Chrysochloris asiatica Gene ID: 102825902, Myotis davidii Gene ID: 102754955, Leptonychotes weddellii Gene ID: 102730427, Lepisosteus oculatus Gene ID: 102692130, Alligator mississippiensis Gene ID: 102576126, Vicugna pacos Gene ID: 102542115, Camelus ferus Gene ID: 102507052, Tupaia chinensis Gene ID: 102482961 , Pelodiscus sinensis Gene ID: 102446147, Myotis lucifugus Gene ID: 102420239,

Bubalus bubalis Gene ID: 102395827, Alligator sinensis Gene ID: 102383307, Latimeria chalumnae Gene ID: 102345318, Pantholops hodgsonii Gene ID: 102326635, Haplochromis burtoni Gene ID: 102295539, Bos mutus Gene ID: 102267392, Xiphophorus maculatus Gene ID: 102228568, Pundamilia nyererei Gene ID: 102192578, Capra hircus Gene ID: 102171407, Pseudopodoces humilis Gene ID: 102106269, Zonotrichia albicollis Gene ID: 102070144, Falco cherrug Gene ID: 102047785, Geospiza fortis Gene ID: 102037409, Chinchilla lanigera Gene ID: 102014610, Microtus ochrogaster Gene ID: 101990242, lctidomys tidecemlineatus Gene ID: 101955193, Chrysemys picta Gene ID: 101939497, Falco peregrinus Gene ID: 101911770, Mesocricetus auratus Gene ID: 101824509, Ficedula albicollis Gene ID: 101814000, Anas platyrhynchos Gene ID: 101789855, Echinops telfairi Gene ID: 101641551 , Condylura cristata Gene ID: 101622847, Jaculus jaculus Gene ID: 101609219, Octodon degus Gene ID: 101563150, Sorex araneus Gene ID: 101556310, Ochotona princeps Gene ID: 101532015, Maylandia zebra Gene ID: 101478751 , Dasypus novemcinctus Gene ID: 101446993, Odobenus rosmarus divergens Gene ID: 101385499, Tursiops truncates Gene ID: 101318662, Orcinus orca Gene ID: 101284095, Oryzias latipes Gene ID: 101154943, Gorilla gorilla Gene ID: 101131184, Ovis aries Gene ID: 101119894, Felis catus Gene ID: 101086577, Takifugu rubripes Gene ID: 101079539, Saimiri boliviensis Gene ID: 101030263, Papio anubis Gene ID: 101004942, Pan paniscus Gene ID: 100981359, Otolemur gamettii Gene ID: 100946205, Sarcophilus harrisii Gene ID: 100928054, Cricetulus griseus Gene ID: 100772179, Cav/a porcellus Gene ID: 100720368, Oreochromis niloticus Gene ID: 100712149, Loxodonta africana Gene ID: 100660074, Nomascus leucogenys Gene ID: 100594138, Anolis carolinensis Gene ID: 100552972, Meleagns gallopavo Gene ID: 100542199, Ailuropoda melanoleuca Gene ID: 100473892, Oryctolagus cuniculus Gene ID: 100339469, Taeniopygia guttata Gene ID: 100225600, Pongo abelii Gene ID: 100172201 , Ornithorhynchus anatinus Gene ID: 100085954, Equus caballus Gene ID: 100052204, Mus musculus Gene ID: 100198, Xenopus laevis Gene ID: 399227, Danio rerio Gene ID: 325181 , Danio rerio Gene ID: 406615, Melopsittacus undulatus Gene ID: 101872435, Ceratotherium simum simum Gene ID: 101408813, Trichechus manatus latirostris Gene ID: 101359849 and Takifugu rubripes Gene ID: 101071719). In the present disclosure, the recombinant microbial cell can reduce or inhibit the expression of a GPD1 gene (or a GPD1 gene ortholog) encoding a GPD1 protein, variant or fragment.

Alternatively or in combination, the genetic modification can include modifying the recombinant host cell to express, in high osmotic conditions, a NAD-dependent glycerol-3-phosphate dehydrogenase 2 (GPD2) protein. This can be done, for example, by expressing a native and/or an heterologous gene (or gene ortholog) encoding the GPD2 protein using an osmotic promoter. In such embodiment, it is important that at least a single native copy of the gene (or the gene ortholog) encoding the GPD2 protein be under the control of the native GPD2 promoter. In the context of the present disclosure, an“osmotic promoter” can be a promoter (or a combination of promoters) allowing the expression (or, in some embodiments, the overexpression) of a gene when the recombinant microbial host cell is placed in high osmotic conditions but refraining the expression (or, in some embodiments, the overexpression) of a gene when the recombinant microbial host cell is placed in normal or low osmotic conditions. In this embodiment, the osmotic promoter can be an inducible promoter . Osmotic promoters are usually associated with genes in the HOG1 pathway and promoters controlling the expression of genes which are upregulated in the HOG1 pathway can be used in the recombinant microbial host cell of the present disclosure. Enzymes in the HOG1 pathway whose expression is upregulated in high osmotic conditions include, but are not limited to, a NAD-dependent glycerol-3-phosphate dehydrogenase 1 gene, a dihydroxyacetone kinase gene and a trehalose- phosphatase gene. As such, in the context of the present disclosure, the osmotic promoter can be a promoter (or a combination of promoters) from a NAD-dependent glycerol-3-phosphate dehydrogenase 1 gene, a dihydroxyacetone kinase gene and/or a trehalose-phosphatase gene. In Saccharomyces cerevisiae, enzymes in the HOG1 pathway whose expression is upregulated in the presence of high osmotic conditions include, but are not limited to, a GPD1 gene, a DAK1 gene and a TPS2 gene. As such, in the context of the present disclosure, the osmotic promoter can be a promoter (or a combination of promoters) from a GPD1 gene (referred to as the GPD1 promoter or gpdl p), a DAK1 gene (referred to as the DAK1 promoter or dak1 p) and/or a TPS2 gene (referred to as the TPS2 promoter or tps2p).

An “osmotic promoter” can also be a constitutive promoter which allows the expression of coding sequences operatively associated thereto during osmotic conditions. In some embodiments, it is preferred that the constitutive promoter be a “low” constitutive promoter. Exemplary “low” constitutive promoters could be associated with the expression of housekeeping genes, and, for example, can include the promoter of the CYC1 gene. In some embodiment, the osmotic promoter is not a high constitutive promoter.

As indicated above, the recombinant microbial host cell can express at least one copy of a native or heterologous GPD protein. In an embodiment, the native or heterologous GPD protein is a native or heterologous GPD2 protein. In the embodiment in which the at least one GPD protein is the GPD2 protein, the recombinant microbial host cell can, in some embodiments, express one, two or more copies of an heterologous gene encoding for the GPD2 protein or a corresponding GPD2 ortholog. When one or more copies of the GPD2 gene or the GPD2 gene ortholog is present in the recombinant microbial host cell, it can be expressed under the control of one or more osmotic promoter(s). In yet a further embodiment, the heterologous GPD2 gene or GPD2 gene ortholog of the recombinant microbial host cell is expressed under the control of the GPD1 promoter, for example, by replacing one or both of the coding sequence of the GPD1 gene by the coding sequence of the GPD2 gene (or the GPD2 gene ortholog).

The GPD2 protein is expressed in bacteria, yeasts, fungi, mammalian and plant cells. GPD2 genes encoding the GPD2 protein include, but are not limited to Mus musculus Gene ID: 14571 , Homo sapiens Gene ID: 2820, Saccharomyces cerevisiae Gene ID: 854095, Rattus norvegicus Gene ID: 25062, Schizosaccharomyces pombe Gene ID: 2541502, Mus musculus Gene ID: 14380, Danio rerio Gene ID: 751628, Caenorhabditis elegans Gene ID: 3565504, Mesocricetus auratus Gene ID: 101825992, Xenopus tropicaiis Gene ID: 779615, Macaca mulatta Gene ID: 697192, Bos taurus Gene ID: 504948, Canis lupus familiaris Gene ID: 478755, Cavia porcellus

Gene ID: 100721200, Gallus gallus Gene ID: 424321 , Pan troglodytes Gene ID: 459670, Oryctolagus cuniculus Gene ID: 100101571 , Candida albicans Gene ID: 3644563, Xenopus laevis Gene ID: 444438, Macaca fascicularis Gene ID: 102127260, Ailuropoda melanoleuca Gene ID: 100482626, Cricetulus griseus Gene ID: 100766128, Heterocephalus glaber Gene ID: 101715967, Scheffersomyces stipitis Gene ID: 4838862, lctalurus punctatus Gene ID:

108273160, Mustela putorius furo Gene ID: 101681209, Nannospalax galili Gene ID: 103741048, Callithrix jacchus Gene ID: 100409379, Lates calcarifer Gene ID: 108873068, Nothobranchius furzeri Gene ID: 07384696, Acanthisitta chloris Gene ID: 103808746, Acinonyx jubatus Gene ID: 106978985, Alligator mississippiensis Gene ID: 102562563, Alligator sinensis Gene ID: 102380394, Anas platyrhynchos, Anolis carolinensis Gene ID: 100551888, Anser cygnoides domesticus Gene ID: 106043902, Aotus nancymaae Gene ID: 105719012, Apaloderma vittatum Gene ID: 104281080, Aptenodytes forsteri Gene ID: 103893867, Apteryx australis mantelli Gene ID: 106486554, Aquila chrysaetos canadensis Gene ID: 105412526, Astyanax mexicanus Gene ID: 103029081 , Austrofundulus limnaeus Gene ID: 106535816, Balaenoptera acutorostrata scammoni Gene ID: 103019768, Balearica regulorum gibbericeps, Bison bison bison Gene ID: 104988636, Bos indicus Gene ID: 109567519, Bos mutus Gene ID: 102277350, Bubalus bubalis Gene ID: 102404879, Buceros rhinoceros silvestris Gene ID: 104497001 , Calidris pugnax Gene ID: 106902763, Callorhinchus milii Gene ID: 103176409, Calypte anna Gene ID: 103535222, Camelus bactrianus Gene ID: 105081921 , Camelus dromedarius Gene ID: 105093713, Camelus ferus Gene ID: 102519983, Capra hircus Gene ID: 102176370, Cariama cristata Gene ID: 104154548, Castor canadensis Gene ID: 109700730, Cebus capucinus imitator Gene ID: 108316996, Cercocebus atys Gene ID: 105576003, Chaetura pelagica Gene ID: 104391744, Charadrius vociferus Gene ID: 104286830, Chelonia mydas Gene ID: 102930483, Chinchilla lanigera Gene ID: 102017931 , Chlamydotis macqueenii Gene ID: 104476789, Chlorocebus sabaeus Gene ID: 103217126, Chrysemys picta Gene ID: 101939831 , Chrysochloris asiatica Gene ID: 102831540, Clupea harengus Gene ID: 105902648, Colius striatus Gene ID: 104549356, Colobus angolensis palliatus Gene ID: 105516852, Columba livia Gene ID: 102090265, Condylura cristata Gene ID: 101619970, Corvus brachyrhynchos, Cotumix japonica Gene ID: 107316969, Crocodylus porosus Gene ID: 109322895, Cuculus canorus Gene ID: 104056187, Cynoglossus semilaevis Gene ID: 103389593, Dasypus novemcinctus Gene ID: 101428842, Dipodomys ordii Gene ID: 105996090, Echinops telfairi Gene ID: 101656272, Egretta garzetta Gene ID: 104135263, Elephantulus edwardii Gene ID: 102858276, Eptesicus fuscus Gene ID: 103283396, Equus asinus Gene ID: 106841969, Equus caballus Gene ID: 100050747, Equus przewalskii Gene ID: 103558835, Erinaceus europaeus Gene ID: 103114599, Eurypyga helias Gene ID: 104502666, Falco cherrug Gene ID: 102054715, Falco peregrinus Gene ID: 101912742, Felis catus Gene ID: 101089953, Ficedula albicollis Gene ID: 101816901 , Fukomys damarensis Gene ID: 104850054, Fundulus heteroclitus Gene ID: 105936523, Galeopterus variegatus Gene ID: 103586331 , Gavia stellata Gene ID: 104250365, Gavialis gangeticus Gene ID: 109301301 , Gekko japonicus Gene ID: 107110762, Geospiza fortis Gene ID: 102042095, Gorilla gorilla Gene ID: 101150526, Haliaeetus albicilla Gene ID: 104323154, Haliaeetus leucocephalus Gene ID: 104829038, Haplochromis burtoni Gene ID: 102309478, Hippocampus comes Gene ID: 109528375, Hipposideros armiger Gene ID: 109379867, lctidomys tridecemlineatus Gene ID: 101965668, Jaculus jaculus Gene ID: 101616184, Kryptolebias marmoratus Gene ID: 108251075, Labrus bergylta Gene ID: 109984158, Larimichthys crocea Gene ID: 104929094, Latimeria chalumnae Gene ID: 102361446, Lepidothrix coronata Gene ID: 108501660, Lepisosteus oculatus Gene ID: 102691231 , Leptonychotes weddellii Gene ID: 102739068, Leptosomus discolor Gene ID: 104340644, Lipotes vexillifer Gene ID: 103074004, Loxodonta afhcana Gene ID: 100654953, Macaca nemestrina Gene ID: 105493221 , Manacus vitellinus Gene ID: 103757091 , Mandrillus leucophaeus Gene ID: 105548063, Manis javanica Gene ID: 108392571 , Marmota marmota marmota Gene ID: 107136866, Maylandia zebra Gene ID: 101487556, Mesitomis unicolor Gene ID: 104545943, Microcebus murinus Gene ID: 105859136, Microtus ochrogaster Gene ID: 101999389, Miniopterus natalensis Gene ID: 107525674, Monodelphis domestica Gene ID: 100014779, Monopterus albus Gene ID: 109957085, Myotis brandtii Gene ID: 102239648, Myotis davidii Gene ID: 102770109, Myotis lucifugus Gene ID: 102438522, Nanorana parked Gene ID: 108784354, Nestor notabilis Gene ID: 104399051 , Nipponia nippon Gene ID: 104012349, Nomascus leucogenys Gene ID: 100590527, Notothenia coriiceps Gene ID: 104964156, Ochotona pnnceps Gene ID: 101530736, Octodon degus Gene ID: 101591628, Odobenus rosmarus divergens Gene ID: 101385453, Oncorhynchus kisutch Gene ID: 109870627, Opisthocomus hoazin Gene ID: 104338567, Orcinus orca Gene ID: 101287409, Oreochromis niloticus Gene ID: 100694147, Ornithorhynchus anatinus Gene ID: 100081433, Orycteropus afer afer Gene ID: 103197834, Oryzias iatipes Gene ID: 101167020, Otolemur garnettii Gene ID: 100966064, Ovis aries Gene ID: 443090, Pan paniscus Gene ID: 100970779, Panthera pardus Gene ID: 109271431 , Panthera tigris altaica Gene ID: 102957949, Pantholops hodgsonii Gene ID: 102323478, Papio anubis Gene ID: 101002517, Paralichthys olivaceus Gene ID: 109631046, Pelodiscus sinensis Gene ID: 102454304, Peromyscus maniculatus bairdii Gene ID: 102924185, Phaethon lepturus Gene ID: 104624271 , Phalacrocorax carbo Gene ID: 104049388, Physeter catodon Gene ID: 102978831 , Picoides pubescens Gene ID: 104296936, Poecilia latipinna Gene ID: 106958025, Poecilia mexicana Gene ID: 106920534, Poecilia reticulata Gene ID: 103473778, Pongo abelii Gene ID: 100452414, Propithecus coquereli Gene ID: 105807399, Protobothrops mucrosquamatus Gene ID: 107289584, Pseudopodoces humilis Gene ID: 102109711 , Pterocles gutturalis Gene ID: 104461236, Pteropus alecto Gene ID: 1028791 10, Pteropus vampyrus Gene ID: 105291402, Pundamilia nyererei Gene ID: 102200268, Pygocentrus nattereri Gene ID: 108411786, Pygoscelis adeliae Gene ID: 103925329, Python bivittatus Gene ID: 103059167, Rhincodon typus Gene ID: 109920450, Rhinolophus sinicus Gene ID: 109445137, Rhinopithecus bieti Gene ID: 108538766, Rhinopithecus roxellana Gene ID: 104654108, Rousettus aegyptiacus Gene ID: 107513424, Saimiri boliviensis Gene ID: 101027702, Salmo salar Gene ID: 106581822, Sarcophilus harrisii Gene ID: 100927498, Scleropages formosus Gene ID: 108927961 , Serinus canaria Gene ID: 103814246, Sinocyclocheilus grahami Gene ID: 107555436, Sorex araneus Gene ID: 101543025, Stegastes partitus Gene ID: 103360018, Struthio camelus australis Gene ID: 104138752, Stumus vulgaris Gene ID: 106861926, Sugiyamaella lignohabitans Gene ID: 30033324, Sus scrofa Gene ID: 397348, Taeniopygia guttata Gene ID: 100222867, Takifugu rubripes Gene ID: 101062218, Tarsius syrichta Gene ID: 103254049, Tauraco erythroiophus Gene ID: 104378162, Thamnophis sirtalis Gene ID: 106538827, Tinamus guttatus Gene ID: 104572349, Tupaia chinensis Gene ID: 102471148, Tursiops truncatus Gene ID: 101330605, Ursus maritimus Gene ID: 103659477, Vicugna pacos Gene ID: 102533941 , Xiphophorus maculatus Gene ID: 102225536, Zonotrichia albicollis Gene ID: 102073261 , Ciona intestinalis Gene ID: 100183886, Meleagris gallopavo Gene ID: 100546408, Trichechus manatus iatirostris Gene ID: 101355771 , Ceratotherium simum simum Gene ID: 101400784, Melopsittacus undulatus Gene ID: 101871704, Esox lucius Gene ID: 10502249 and Pygocentrus nattereri Gene ID: 10841 1786. In an embodiment, the GPD2 protein is encoded by Saccharomyces cerevisiae Gene ID: 854095.

The heterologous GPD2 protein can be encoded by a GPD2 gene or a GPD2 gene ortholog as defined herein. The heterologous GPD2 protein can also be a variant of the GPD2 protein and/or a fragment of the GPD2 protein. In addition, when more than one copy of the heterologous GPD2 gene or gene ortholog is included in the recombinant microbial cell, the plurality of heterologous nucleic acid molecules encoding the GPD2 protein could be the same or different, integrated at the same or different integration sites. In some embodiments, the recombinant microbial host cell can include one or more additional genetic modifications coding for an enzyme, can be co-cultured with additional recombinant host cells including additional genetic modifications coding for enzymes or can be used with heterologous (purified) enzymes described herein.

For example, the additional enzyme can allow for the production of an heterologous glucoamylase. Many microbes produce an amylase to degrade extracellular starches. In addition to cleaving the last a(1 - 4) glycosidic linkages at the non-reducing end of amylose and amylopectin, yielding glucose, g-amylase will cleave a(1 -6) glycosidic linkages. The heterologous glucoamylase can be derived from any organism. In an embodiment, the heterologous protein is derived from a g-amylase, such as, for example, the glucoamylase of Saccharomycoces filbuligera (e.g., encoded by the glu 01 11 gene). Examples of recombinant yeast host cells expressing such enzymes are described in WO 2011/153516 as well as in WO 2017/037614 filed on August 29, 2016.

In yet another example, the enzyme can reduce the production of one or more native enzyme that function to catabolize (breakdown) formate. As used in the context of the present disclosure, the expression “native polypeptides that function to catabolize formate” refers to polypeptides which are endogenously found in the recombinant yeast host cell. Native enzymes that function to catabolize formate include, but are not limited to, the FDH1 and the FDH2 polypeptides (also referred to as FDH1 and FDH2 respectively). In an embodiment, the recombinant yeast host cell bears a genetic modification in at least one of the FDH1 gene (encoding the FDH1 polypeptide), the FDH2 gene (encoding the FDH2 polypeptide) or orthologs thereof. In another embodiment, the recombinant yeast host cell bears genetic modifications in both the FDH1 gene (encoding the FDH1 polypeptide) and the fdh2 gene (encoding the FDH2 polypeptide) or orthologs thereof. Examples of recombinant yeast host cells bearing such genetic modification(s) leading to the reduction in the production of one or more native enzymes that function to catabolize formate are described in WO 2012/138942. Preferably, the recombinant yeast host cell has genetic modifications (such as a genetic deletion or insertion) in the FDH1 gene and in the FDH2 gene which would cause the host cell to have knocked-out FDH1 and FDH2 genes.

In still another example, the enzyme can increase the production of an heterologous enzyme that function to anabolize (form) formate. As used in the context of the present disclosure,“an enzyme that functions to anabolize formate” refers to polypeptides which may or may not be endogeneously found in the recombinant yeast host cell and that are purposefully introduced into the recombinant yeast host cells. In some embodiments, the heterologous enzyme that function to anabolize formate is an heterologous pyruvate formate lyase (PFL), an heterologous acetaldehyde dehydrogenases, an heterologous alcohol dehydrogenases, and/or and heterologous bifunctional acetylaldehyde/alcohol dehydrogenases (AADH) such as those described in US Patent Serial Number 8,956,851 and WO 2015/023989. More specifically, PFL and AADH enzymes for use in the recombinant yeast host cells can come from a bacterial or eukaryotic source. Heterologous PFL of the present disclosure include, but are not limited to, the PFLA polypeptide, a polypeptide encoded by a PFLA gene ortholog, the PFLB polyeptide or a polypeptide encoded by a PFLB gene ortholog. Heterologous MDHs of the present disclosure include, but are not limited to, the ADHE polypeptides or a polypeptide encoded by an ADHE gene ortholog. In an embodiment, the recombinant yeast host cell of the present disclosure comprises at least one of the following heterologous enzymes that function to anabolize formate: the PFLA polypeptide, the PFLB polypeptide and/or the ADHE polypeptide. In an embodiment, the recombinant yeast host cell of the present disclosure comprises at least two of the following heterologous enzymes that function to anabolize formate: the PFLA polypeptide, the PFLB polypeptide and/ or the ADHE polypeptide. In another embodiment, the recombinant yeast host cell of the present disclosure comprises the following heterologous enzymes that function to anabolize formate: the PFLA polypeptide, the PFLB polypeptide and the ADHE polypeptide.

In some embodiments, the enzyme involved in the cleavage or hydrolysis of its substrate (e.g., a lytic enzyme and, in some embodiments, a saccharolytic enzyme). In still another embodiment, the enzyme can be a glycoside hydrolase. In the context of the present disclosure, the term “glycoside hydrolase” refers to an enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, trehalases, pectinases, dextranases, and pentose sugar utilizing enzymes. In another embodiment, the enzyme can be a protease. In the context of the present disclosure, the term“protease” refers to an enzyme involved in protein digestion, metabolism and/or hydrolysis. In yet another embodiment, the enzyme can be an esterase. In the context of the present disclosure, the term“esterase” refers to an enzyme involved in the hydrolysis of an ester from an acid or an alcohol, including phosphatases such as phytases.

The additional enzyme can be an “amylolytic enzyme", an enzyme involved in amylose digestion, metabolism and/or hydrolysis. The term“amylase” refers to an enzyme that breaks starch down into sugar. All amylases are glycoside hydrolases and act on a-1 ,4-glycosidic bonds. Some amylases, such as g-amylase (glucoamylase), also act on a-1 ,6-glycosidic bonds. Amylase enzymes include a-amylase (EC 3.2.1 .1), b-amylase (EC 3.2.1.2), and g-amylase (EC 3.2.1.3). The a-amylases are calcium metalloenzymes, unable to function in the absence of calcium. By acting at random locations along the starch chain, a-amylase breaks down long- chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and“limit dextrin” from amylopectin. Because it can act anywhere on the substrate, a- amylase tends to be faster-acting than b-amylase. In an embodiment, the heterologous protein is derived from a a-amylase such as, for example, from the a-amylase of Bacillus amyloliquefacens. Another form of amylase, b-amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, b-amylase catalyzes the hydrolysis of the second a-1 ,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Another amylolytic enzyme is a-glucosidase that acts on maltose and other short malto-oligosaccharides produced by a-, b-, and g-amylases, converting them to glucose. Another amylolytic enzyme is pullulanase. Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan is regarded as a chain of maltotriose units linked by alpha- 1 ,6- glycosidic bonds. Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase (debranching enzyme). Another amylolytic enzyme, isopullulanase, hydrolyses pullulan to isopanose (6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known as pullulan 4- glucanohydrolase. An“amylase” can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis, including a-amylase, b -amylase, glucoamylase, pullulanase, isopullulanase, and alpha-glucosidase.

The additional enzyme can be a dextranase. Dextran is a complex branched polysaccharide composed of glucose monomer units. It contains a straight chain of a-1 ,6 glycosidic linkages, and branches linked by a-1 ,2, a-1 ,3, or a-1 ,4 glycosidic bonds. Several enzymes participate in the breakdown of dextran. Dextranase (EC 3.2.1 .1 1), also known as alpha-1 ,6-glucan-6- glucanohydrolase, is an enzyme that carries out the endohydrolysis of a-1 ,6 glycosidic bonds in dextran. Other enzymes that act to break down dextran include: glucan-1 ,6-a-D-glucosidases (EC3.2.1.70), glucan-1 ,6-a-isomaltosidases (EC3.2.1.94), dextran 1 ,6-a-isomaltotriosidases (EC3.2.1.95), branched-dextran exo-1 ,2-a-glucosidases (EC3.2.1 .1 15), a-glucosidase (EC3.2.1.20), and Cycloisomaltooligosaccharide glucanotransferase (CITase).

The additional enzyme can be a “cellulolytic enzyme”, an enzyme involved in cellulose digestion, metabolism and/or hydrolysis. The term“cellulase” refers to a class of enzymes that catalyze cellulolysis (i.e. the hydrolysis) of cellulose. Several different kinds of cellulases are known, which differ structurally and mechanistically. There are general types of cellulases based on the type of reaction catalyzed: endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains; exocellulase cleaves 2-4 units from the ends of the exposed chains produced by endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose. There are two main types of exocellulases (or cellobiohydrolases, abbreviate CBH) - one type working processively from the reducing end, and one type working processively from the non- reducing end of cellulose; cellobiase or beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides; oxidative cellulases that depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor); cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. In the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to beta-glucose. A“cellulase” can be any enzyme involved in cellulose digestion, metabolism and/or hydrolysis, including an endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase, acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin, pectinase, and feruoyl esterase protein.

The additional enzyme can have“hemicellulolytic activity”, an enzyme involved in hemicellulose digestion, metabolism and/or hydrolysis. The term“hemicellulase" refers to a class of enzymes that catalyze the hydrolysis of cellulose. Several different kinds of enzymes are known to have hemicellulolytic activity including, but not limited to, xylanases and mannanases.

The additional enzyme can have “xylanolytic activity", an enzyme having the is ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses. The term“xylanase” is the name given to a class of enzymes which degrade the linear polysaccharide beta-1 ,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Xylanases include those enzymes that correspond to Enzyme Commission Number 3.2.1.8. The heterologous protein can also be a“xylose metabolizing enzyme”, an enzyme involved in xylose digestion, metabolism and/or hydrolysis, including a xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, and a xylose transaldolase protein. A“pentose sugar utilizing enzyme” can be any enzyme involved in pentose sugar digestion, metabolism and/or hydrolysis, including xylanase, arabinase, arabinoxylanase, arabinosidase, arabinofuranosidase, arabinoxylanase, arabinosidase, and arabinofuranosidase, arabinose isomerase, ribulose-5-phosphate 4- epimerase, xylose isomerase, xylulokinase, xylose reductase, xylose dehydrogenase, xylitol dehydrogenase, xylonate dehydratase, xylose transketolase, and/or xylose transaldolase.

The additional enzyme can have “mannanic activity”, an enzyme having the is ability to hydrolyze the terminal, non-reducing b-D-mannose residues in b-D-mannosides. Mannanases are capable of breaking down hemicellulose, one of the major components of plant cell walls. Xylanases include those enzymes that correspond to Enzyme Commission Number 3.2.25.

The additional enzyme can be a“pectinase”, an enzyme, such as pectolyase, pectozyme and polygalacturonase, commonly referred to in brewing as pectic enzymes. These enzymes break down pectin, a polysaccharide substrate that is found in the cell walls of plants.

The additional enzyme can have“phytolytic activity”, an enzyme catalyzing the conversion of phytic acid into inorganic phosphorus. Phytases (EC 3.2.3) can be belong to the histidine acid phosphatases, b-propeller phytases, purple acid phosphastases or protein tyrosine phosphatase-like phytases family. The additional enzyme can have“proteolytic activity”, an enzyme involved in protein digestion, metabolism and/or hydrolysis, including serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.

When the recombinant yeast host cell expresses a heterologous protein, it can be further modified to increase its robustness at high temperatures. Genetic modifications for increasing the robustness of a genetically-modified recombinant yeast host cell are described in PCT/IB2016/055162 filed on August 29, 2016.

In some embodiments, the recombinant microbial host cells of the present disclosure do not have (e.g., exclude) a genetic modification in its NADH-consuming glutamate synthase gene. In Saccharomyces cerevisiae, the NADH-consuming glutamate synthase gene is known as GLT1 (as described in Wang et al., 2013).

In still another embodiment, the recombinant microbial host cells of the present disclosure does not (e.g., exclude) genetic modifications in genes encoding heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to an alcohol, such as those described in WO2015/023989.

Preventing or limiting microbial contamination during continuous cultures

The present disclosure also provides a process for preventing or limiting microbial contamination during a continuous culture. As indicated, a “continuous culture” of the recombinant yeast host cell refers to a culture lasting at least a fermentation cycle and is subject to take-over from wild-type contaminating microorganisms, such as, for example, by wild-type yeasts. Continuous cultures are performed, for example, in the Brazilian biofuel processes as well as in other long running ethanol process. Contaminating microorganisms causing the microbial contamination include, but are not limited to, contaminating yeasts (such as, for example, from the Saccharomyces sp. (e.g., Saccharomyces cerevisiae ), the Dekkera sp. (e.g., Dekkera bruxellensis ), the Candida sp. (e.g., Candida krusei) and/or the Schizosaccharomyces sp. (e.g., Schizosaccharomyces pombe) as well as bacteria. In an embodiment, the contaminating microorganisms are yeasts. In yeast another embodiment, the contaminating microorganisms comprise a majority of yeasts from the Saccharomyces sp. and a minority of yeasts from the non- Saccharomyces sp. (such as, for example, from the Dekkera sp., the Candida sp. and/or the Schizosaccharomyces sp.). In yet another embodiment, the contaminating microorganisms comprise at least 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95% or more of yeasts from the Saccharomyces sp.. In still another embodiment, the contaminating microorganisms comprise no more than 40%, 30%, 20%, 10%, 5% or less of yeasts from the non- Saccharomyces sp.

In its broadest embodiment, the process comprises culturing the recombinant yeast host cell in the presence of the pro-cytotoxic agent. The pro-cytotoxic agent can be included in the medium of continuous culture prior to the culture of the recombinant yeast host cell (e.g., in a prophylactic manner). For example, the pro-cytotoxic agent can be included in the (sugar cane) must prior to fermentation. Alternatively or complementarily, the cytotoxic agent can be contacted with the recombinant yeast host cell during the continuous culture. The process can also include contacting once or at a plurality of occasions the pro-cytotoxic agent with the recombinant yeast host cell during the continuous culture. The process can further comprise adding the pro-cytotoxic agent to the medium of the continuous culture, before and/or during the culture of the recombinant yeast host cell.

In one embodiment, the process described herein provides for limiting its extent of the microbial contamination between 0.1-10% of the total microbial population of the medium (or in a sample thereof, as measured by CFU or the total number of cells). In an embodiment, the process can be used to limit the microbial contamination to levels equal to or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or 0.2% with respect to the total microbial population of the medium (or in a sample thereof). In another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% with respect to the total microbial population of the medium (or in a sample thereof). In yet another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9% with respect to the total microbial population of the medium (or in a sample thereof). In still another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7% or 8% with respect to the total microbial population of the medium (or in a sample thereof). In yet another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6% or 7% with respect to the total microbial population of the medium (or in a sample thereof). In another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5% or 6% with respect to the total microbial population of the medium (or in a sample thereof). In still another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4% or 5% with respect to the total microbial population of the medium (or in a sample thereof). In a further embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3% or 4% with respect to the total microbial population of the medium (or in a sample thereof). In yet another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2% or 3% with respect to the total microbial population of the medium (or in a sample thereof). In another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 % or 2% with respect to the total microbial population of the medium (or in a sample thereof). In yet another embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1 % with respect to the total microbial population of the medium (or in a sample thereof). In a further embodiment, the process can be used to limit the microbial contamination to levels between about 0.1 % and 1 % with respect to the total microbial population of the medium (or in a sample thereof). In continuous cultures, such as for example in the Brazilian fuel ethanol fermentations, microbial contamination can cause unfavorable fermentation condition (foaming and flocculation for example) as well as a decreased productivity. Therefore by limiting the presence of the microbial contamination to a certain level, an increase in productivity can be observed.

The process of the present disclosure can comprise alternating between two media to limit the microbial contamination: a first medium lacking a pro-cytotoxic agent and a second medium having the pro-cytotoxic agent. The process can be undertaken with a first medium lacking the pro-cytotoxic agent or a second medium comprising the pro-cytotoxic agent. Even though, a reference is made to a “first” medium and a “second” medium, the present disclosure contemplated using any medium before the other. In an embodiment, the second medium is used prior to the first medium and vice versa.

The pro-cytotoxic agent to be used in the process is selected in function of the recombinant yeast host cell used in such process and more specifically in function of the genetic modification that has been made to the recombinant yeast host cell. As indicated herein, the recombinant yeast host cell includes at least one genetic modification to decrease or impede the expression of at least one noxious gene. Such noxious gene, present and expressed in the contaminating microorganism(s), encodes a polypeptide capable of metabolizing the pro-cytotoxic agent (which lacks cytotoxicity towards the recombinant yeast host cell(s) and the contaminating microorganism(s)) into a cytotoxic agent (which is cytotoxic towards the contaminating microorganism(s) but not the recombinant yeast host cell(s)). As such, the type of pro-cytotoxic agent used is based on the genetic modification(s) of the recombinant yeast host cell present in the medium. For example, when the first recombinant yeast host cell has been genetically modified so as not to express the FCY1 gene and/or the FUR1 gene (or their corresponding gene orthologs), the pro-cytotoxic agent can be 5-FC and/or 5-FU.

In an embodiment, the process described herein provides alternating between the two medium in function of a first (maximal) threshold of microbial contamination and a second (minimal) threshold of microbial contamination. The first threshold is used so as to limit the microbial contamination prior its take-over of the culture. The second threshold is used so as to limit the use/costs of the pro-cytotoxic agent. The culture can be started in the absence of the pro- cytotoxic agent, even though it is possible at this stage of the culture to include the pro-cytotoxic agent in the medium (especially if the culture is a repetition of numerous fermentation cycles and the previous fermentation cycle include an important microbial contamination, e.g., above or close to the first threshold). Alternatively, the culture can be started in the presence of the pro-cytotoxic agent, even though it is possible at this stage of the culture to exclude the pro- cytotoxic agent in the medium (especially if the culture is a repetition of numerous fermentation cycles and the previous fermentation cycle include a minimal microbial contamination, e.g., lower to the second threshold). Because the process described herein is intended to be used during continuous cultures which are expected to be contaminated with contaminating microorganisms, when the first threshold of microbial contamination is exceeded, the process provides, in some embodiments, culturing the recombinant yeast host cell in the second medium comprising the pro-cytotoxic agent. The recombinant yeast host cell can be cultured in the presence of the pro-cytotoxic agent until the second threshold is reached. At that point, the recombinant yeast host cell can be cultured in the absence of the pro-cytotoxic agent until the first threshold is exceeded again.

Thus, the process can comprise culturing the recombinant yeast host cell in a first medium lacking the pro-cytotoxic agent, until the first threshold of microbial contamination is exceeded. As indicated above, if the first threshold of microbial contamination is excess at the onset of the culture or during the initial propagation step of the culture, the process can include culturing the recombinant yeast host cell in the second medium in the presence of the pro-cytotoxic agent. Further, if the first threshold is never exceeded during the culture, it is contemplated that the recombinant yeast host cell can be cultured only in the absence of the pro-cytotoxic agent. The first threshold corresponds to a contamination level that can be controlled by the addition of the cytotoxic agent. In an embodiment, the first threshold is above about 0.1 % of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.2% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.3% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.4% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.5% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.6% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.7% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.8% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 0.9% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 1 % of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 2% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 3% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 4% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 5% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 6% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 7% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 8% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 9% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In an embodiment, the first threshold is above about 10% of contaminating microorganisms when compared to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 0.1-1 %. 0.1-10%, 0.1-9%, 0.1-8%, 0.1-7%, 0.1 -6%, 0.1-5%, 0.1-4%, 0.1 -3% or 0.1-2% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 2-10%, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2-4%, or 2-3% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 3-10%, 3-9%, 3-8%, 3-7%, 3-6%, 3-5% or 3-4% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 4- 10%, 4-9%, 4-8%, 4-7%, 4-6% or 4-5% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 5-10%, 5-9%, 5-8%, 5-7% or 5-6% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 6-10%, 6-9%, 6-8% or 6-7% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 7-10%, 7-9% or 7-8% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 8-10% or 8-9% with respect to the total microbial population of the medium or the sample thereof. In yet another embodiment, the first threshold corresponds to between about 9-10% with respect to the total microbial population of the medium or the sample thereof. In still another embodiment, the first threshold corresponds to between about 0.1-1 % with respect to the total microbial population of the medium or the sample thereof.

The process can also comprise culturing the recombinant yeast host cell in a second medium comprising the pro-cytotoxic agent until a second threshold of microbial contamination is reached. The second threshold of microbial contamination is necessarily lower than the first threshold of contamination. If the second threshold of microbial contamination is not reached during the culture, the process provides culturing the recombinant yeast host cell(s) in the presence of the cytotoxic agent during the entire length of the culture. As such, in some embodiments, the process provides culturing the recombinant yeast host cells only in a medium comprising the pro-cytotoxic agent. The second threshold corresponds to a microbial contamination level equal to or less than about 0.1 %, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01 % or lower with respect to the total microbial population of the medium or a sample thereof. In some embodiments, a microbial contamination level equal to or less than about 0.1 % with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.09% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.08% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.07% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.06% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.05% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.04% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.03% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.02% with respect to the total microbial population of the medium or a sample thereof. In other embodiments, the second threshold corresponds to a microbial contamination level equal to or less than about 0.01 % with respect to the total microbial population of the medium or a sample thereof.

The steps described herein can be repeated more than once and as such the recombinant yeast host cell can alternate, during the entire course of the continuous culture, between a medium comprising and lacking a pro-cytotoxic agent, depending on the level of microbial contamination. In an example, the steps of the process described herein can be repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , or 52 times a year. In some embodiments, the steps of the process described herein can be repeated once, twice or thrice a year.

An embodiment of the process of the present disclosure is provided in Figure 1 . In the embodiment of Figure 1 , a continuous culture is provided and, at step 010, the total microbial population is cultured in the absence of the pro-cytotoxic agent and is being monitored for the presence and the extent of a contamination by contaminating microorganisms. As used in the context of the present disclosure, a step of monitoring refers to a step that is performed more than once at two different time point. The monitoring step can inform if a microbial contamination is present, absent, increasing, persisting or declining. Monitoring can be done, for example, by assessing, the number of contaminating microorganisms, the percentage or ratio of contaminating microorganisms in function of the total microbial population of the cultured medium (or a sample thereof). This can also be done by assessing the number of recombinant yeast host cells, the percentage or ratio of recombinant yeast host cells in function of the total microbial population of the cultured medium (or a sample thereof). In the embodiment shown on Figure 1 , it is the percentage of contaminating microorganisms in function of the total microbial population of the cultured medium or the sample thereof that is being monitored. Monitoring can be performed many times during the continuous culture process, for example, it can be done on a monthly or on a weekly basis. If the monitoring step 010 provides that a microbial contamination is present in the cultured medium and that this microbial contamination exceeds a first threshold (for example 10% in the embodiment shown on Figure 1), then the process includes a step 020 of culturing the recombinant yeast host cell(s) in the presence of the pro- cytotoxic agent in an effort to reduce the microbial contamination. If the monitoring step 010 provides that a microbial contamination is present in the cultured medium and that this microbial contamination is equal to or lower than a first threshold (for example 10% in the embodiment shown on Figure 1), then the recombinant yeast host cell(s) continues to be cultured in the absence of the pro-cytotoxic agent. It is understood that, if at the start or the initial stages of the culture, it is determined that the level of microbial contamination exceeds the first threshold (for example when a microbial contamination determination is made on the starting culture), the starting medium of the culture can include the pro-cytotoxic agent (not shown on Figure 1). Once the recombinant yeast host cell is cultured in the presence of pro-cytotoxic agent, the medium is further monitored, at step 030, to determine the presence and the extent of a contamination by contaminating microorganisms until a second threshold is reached (for example 0.1 % on the embodiment shown on Figure 1). Monitoring can be performed many times during the continuous culture process, for example, it can be done on a monthly or on a weekly basis. If the monitoring step 030 provides that a microbial contamination is present in the cultured medium and that this microbial contamination is lower than a second threshold (for example 0.1 % in the embodiment shown on Figure 1), then the process includes a step 040 of culturing the recombinant yeast host cell(s) in the absence of the pro-cytotoxic agent. On the other hand, if the monitoring step 030 provides that a microbial contamination is present in the cultured medium and that this microbial contamination exceeds a second threshold (for example 0.1 % in the embodiment shown on Figure 1), then the process provides for continuing culturing, at step 020, the recombinant yeast host cell(s) in the presence of the pro-cytotoxic agent. It is understood that, if any time of the culture, it is determined that the level of microbial contamination is equal or lower than the second threshold, the recombinant yeast host cells is to be cultured in a medium lacking the pro-cytotoxic agent (not shown on Figure 1).

It will be understood that the process shown on Figure 1 is a cyclic approach and that the process described herein can start at any steps.

In some alternative embodiments, the process can comprise culturing the recombinant yeast host cell in the presence of the pro-cytotoxic agent throughout the continuous culture so as to prevent a microbial contamination.

In the present disclosure, a recombinant yeast host cell having a genetic modification causing the reduction or the inhibition of the expression the noxious gene FCY1 can be used in the processes in combination with 5-fluorocytosine (5-FC) and/or 5-fluorouracil (5-FU) as the pro- cytotoxic agent. Optionally, the recombinant yeast host cell can further include a further genetic modification causing the reduction or the inhibition of the expression the noxious gene FUR1 . Alternatively, a recombinant yeast host cell having a genetic modification causing the reduction or the inhibition of the expression the noxious gene FUR1 can be used in the processes in combination with 5-fluorocytosine (5-FC) and/or 5-fluorouracil (5-FU) as the pro-cytotoxic agent.

In an embodiment of the process, a continuous culture can be provided and the total microbial population is being monitored, for example monthly or weekly, for the presence and the extent of a contamination by contaminating microorganisms. In such embodiment shown, it is the percentage of contaminating microorganisms in function of the total microbial population of the cultured medium or the sample thereof that is being monitored. If the monitoring step provides that a microbial contamination is present in the cultured medium and that this microbial contamination exceeds a first threshold (for example 10%), then the process includes a step of culturing the recombinant yeast host cell(s) in the presence of the 5-FC/5-FU in an effort to reduce the microbial contamination. Since, in an embodiment, the recombinant yeast host cells do not express the FCY1 gene and/or the FUR1 gene (or their corresponding orthologs), they cannot metabolize 5-FC/5-FU into a cytotoxic compound and can continue to grow. In addition, since the contaminating microorganisms, especially if they are contaminating yeasts, express the FCY1 gene and/or the FUR1 gene (or their corresponding orthoglos) which codes for the FCY1 protein and/or the FUR1 protein, are capable of metabolizing 5-FC/5-FU into a cytotoxic compound, their growth or viability will be limited. In an embodiment, the process comprises adding to the first medium at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium no more than about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising no more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 10 to 500 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 10 to 500 ppm of 5-FC/5-FU.

Once the recombinant yeast host cell is cultured in the presence of pro-cytotoxic agent, the medium is further monitored to determine the presence and the extent of a contamination by contaminating microorganisms until a second threshold is reached (for example 0.1 %). If the monitoring step provides that a microbial contamination is present in the cultured medium and that this microbial contamination is lower than a second threshold (for example 0.1 %), then the process includes a step of culturing the recombinant yeast host cell(s) in the absence of 5-FC/5- FU. On the other hand, if the monitoring step provides that a microbial contamination is present in the cultured medium and that this microbial contamination exceeds a second threshold (for example 0.1 %), then the process includes continuing culturing the recombinant yeast host cell(s) in the presence of 5-FC/5-FU.

Processes of making a fermented product

The process of limiting microbial contamination described herein can be specifically applied to processes for making fermented products from a substrate. In such embodiment, the medium of the continuous culture comprises a carbohydrate source (e.gr., a first and/or second medium comprising a carbohydrate) allowing the production of fermented products. During the culture, the first and/or second medium is being fermented by the recombinant yeast host cell to make a fermented product. The fermented product can be an alcohol, such as, for example, ethanol, isopropanol, n-propanol, 1 -butanol, methanol, acetone, 1 , 2 propanediol or an heterologous polypeptide that is expressed in a recombinant fashion by the recombinant yeast host cell. In its broadest embodiment, the process comprising culturing the recombinant yeast host cell in the presence of the pro-cytotoxic agent. The pro-cytotoxic agent can be included in the first and/or second medium prior to the fermentation process. Alternatively or complementarily, the cytotoxic agent can be contacted with the recombinant yeast host cell prior to or during the continuous fermentation process. The process can also include contacting once or at a plurality of occasions the pro-cytotoxic agent with the recombinant yeast host cell during the fermentation process. The process can further comprise adding the pro-cytotoxic agent to the first medium before and/or during the fermentation process.

In some embodiments of the fermentation process, the recombinant yeast host cells are submitted to continuous cultures which is susceptible from being taken-over by wild-type contaminating microorganisms, such as, for example, by wild-type yeasts. Such fermentations include, but are not limited to, the Brazilian biofuel processes as well as in other long running ethanol process. As indicated herein, contaminating microorganisms causing the microbial contamination include, but are not limited to, contaminating yeasts and bacteria.

In an embodiment, during the fermentation, and as described herein, the process can comprise alternating between two fermentation media to limit the microbial contamination: a first fermentation medium comprising a carbohydrate and lacking a pro-cytotoxic agent and a second fermentation medium comprising a carbohydrate and having the pro-cytotoxic agent. The pro-cytotoxic agent to be used in the process is selected in function of the recombinant yeast host cell used in such process and more specifically in function of the genetic modification that has been made to the recombinant yeast host cell. As indicated herein, the recombinant yeast host cell includes at least one genetic modification to decrease or impede the expression of at least one noxious gene. Such noxious gene, present and expressed in the contaminating microorganism(s), encodes a polypeptide capable of metabolizing the pro-cytotoxic agent (which lacks cytotoxicity towards the recombinant yeast host cell(s) and the contaminating microorganism(s)) into a cytotoxic agent (which is cytotoxic towards the contaminating microorganism(s) but not the recombinant yeast host cell(s)). As such, the type of pro-cytotoxic agent used is based on the genetic modification(s) of the recombinant yeast host cell present in the fermentation medium. For example, when the first recombinant yeast host cell has been genetically modified so as not to express the FCY1 gene and/or the FUR1 gene (or their corresponding orthologs), the pro-cytotoxic agent can be 5-FC and/or 5-FU.

In an embodiment, the process provides alternating between the two fermentation media in function of a first (maximal) threshold of microbial contamination and a second (minimal) threshold of microbial contamination as described above. The first threshold is used so as to limit the microbial contamination prior its take-over of the culture. The second threshold is used so as to limit the use/costs of the pro-cytotoxic agent. The fermentation can be started in the absence of the pro-cytotoxic agent, even though it is possible at this stage of the fermentation to include the pro-cytotoxic agent in the fermentation medium (especially if the culture is a repetition of numerous fermentation cycles and the previous fermentation cycle included an important microbial contamination, e.g., above or close to the first threshold). Because the process described herein is intended to be used during continuous fermentations and is expected to be contaminated with contaminating microorganisms, when the first threshold of microbial contamination is exceeded, the process provides fermenting the recombinant yeast host cell in the second fermentation medium comprising the pro-cytotoxic agent. The recombinant yeast host cell ferments the second fermentation medium in the presence of the pro-cytotoxic agent until the second threshold is reached. At that point, the recombinant yeast host cell can be cultured in the absence of the pro-cytotoxic agent until the first threshold is reached.

In some embodiments, and as indicated herein for the continuous culture process, the fermentation process described herein provides for limiting its extent of the microbial contamination between 0.1 to 10% of the total microbial population of the medium (or in a sample thereof). For example, the recombinant yeast host cell can be cultured in a first (fermentation) medium until a first contamination threshold is exceeded and, in some embodiments, in a second (fermentation) medium until a second contamination threshold is reached. Alternatively, the recombinant yeast host cell can be cultured in a second (fermentation) medium until a second contamination threshold is reached and, in some embodiments, in a first (fermentation) medium until a first contamination threshold is exceeded. The first contamination threshold is necessarily higher than the second contamination threshold. The first threshold can be used so as to limit the microbial contamination prior its take-over of the culture. The second threshold can be used so as to limit the use/costs of the pro-cytotoxic agent. The first contamination threshold can be, as indicated herein, between about 1 to 10%. The second contamination threshold can be, as indicated herein, equal to or less than about 0.1 %.

The steps described herein can be repeated more than once and as such the recombinant yeast host cell can alternate, during the entire course of the fermentation, between a medium comprising and lacking a pro-cytotoxic agent, depending on the level of microbial contamination. In an example, the steps of the process described herein can be repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , or 52 times a year. In some embodiments, the steps of the process described herein can be repeated once, twice or thrice a year. The embodiment of the process shown on Figure 1 can be applied to the fermentation process of the present disclosure, provided that the first and/or second medium comprise a carbohydrate source. As indicated herein for the continuous culture process, the fermentation process can also be submitted to a monitoring step to determine the presence and the extend of a contamination by contaminating microorganisms.

In the present disclosure, a recombinant yeast host cell having a genetic modification causing the reduction or the inhibition of the expression the noxious gene FCY1 and/or FUR 1 (or their corresponding orthologs) can be used in the fermentation processes in combination with 5-FC and/or 5-FU as the pro-cytotoxic agent. In an embodiment, a fermentation can be provided and the total microbial population can be being monitored, for example monthly or weekly, for the presence and the extent of a contamination by contaminating microorganisms. In such embodiment shown, it is the percentage of contaminating microorganisms in function of the total microbial population of the fermentation medium or the sample thereof that is being monitored. If the monitoring step provides that a microbial contamination is present in the fermentation medium and that this microbial contamination exceeds a first threshold (for example 10%), then the process includes a step of culturing the recombinant yeast host cell(s) in the presence of the 5-FC/5-FU in an effort to reduce the microbial contamination. Since the recombinant yeast host cells do not express the FCY1 gene and/or the FUR1 gene (or their corresponding orthologs), they cannot metabolize 5-FC/5-FU and can continue to grow. In addition, since the contaminating microorganisms, especially if they are contaminating yeasts, express the FCY1 gene and the FUR1 gene (or their corresponding orthologs) which codes for the FCY1 protein and the FUR1 protein are capable of metabolizing 5-FC/5-FU into a cytotoxic compound, their growth or viability will be limited. In an embodiment, the process comprises adding to the first medium at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU to provide the second medium. For example, the process can comprise adding to the first medium at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ppm of %- FC/5-FU. In still another example, the process can comprise adding between about 0.1 and 10 ppm to the first medium to obtain the second medium. In another embodiment, the process comprises providing a second medium comprising at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU. For example, the process can comprise providing a second medium comprising at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ppm. In still another example, the process can comprise providing a second medium comprising between about 0.1 and 10 ppm 5-FC/5-FU.

In an embodiment, the process comprises adding to the first medium no more than about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises adding to the first medium no more than about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ppm 5-FC/5-FU to obtain the second medium. In another embodiment, the process comprises providing a second medium comprising no more than about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 ppm of 5-FC/5-FU. In yet another embodiment, the process comprises providing a second medium comprising no more than about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 0.1 to 500 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 0.1 to 500 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 0.1 to 10 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 0.1 to 10 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 0.1 to 5 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 0.1 to 5 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 0.1 to 3 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 0.1 to 3 ppm of 5-FC/5-FU. In an embodiment, the process comprises adding to the first medium between about 0.2 to 3 ppm of 5-FC/5-FU to provide the second medium. In another embodiment, the process comprises providing a second medium comprising between about 0.2 to 30 ppm of 5-FC/5-FU.

Once the recombinant yeast host cell is cultured in the presence of pro-cytotoxic agent, the medium can be further monitored to determine the presence and the extent of a contamination by contaminating microorganisms until a second threshold is reached (for example 0.1 %). If the monitoring step provides that a microbial contamination is present in the fermentation medium and that this microbial contamination is lower than a second threshold (for example 0.1 %), then the process includes a step of culturing the recombinant yeast host cell(s) in the absence of 5- FC. On the other hand, if the monitoring step provides that a microbial contamination is present in the cultured medium and that this microbial contamination exceeds a second threshold (for example 0.1 %), then the process includes continuing culturing the recombinant yeast host cell(s) in the presence of 5-FC/5-FU.

The genetic modification of the recombinant yeast host cell selected to conduct the fermentation preferably does not substantially alter the fermentation performance (such as, for example, the fermentation rate, the growth, the viability and/or the robustness) of the recombinant yeast host cell when compared to the fermentation performance of a corresponding yeast host cell lacking the genetic modification. As used in the context of the present disclosure, the fermentation performance of the recombinant yeast host cells is not substantially altered (and in some embodiments, it may be increased) when compared to the fermentation performances of a corresponding host cell lacking the genetic modification. As used in the context of the present disclosure, the term “is not substantially altered” refers to the fact that the fermentation performances of the recombinant yeast host cell can be reduced by at most 1 , 2, 5, 10, 15, 20 or 25% when compared to the fermentation performances of the corresponding host cell lacking the genetic modification. The performance during fermentation of a yeast cell can be measured by determining the fermentation rate (the higher the fermentation rate, the better the performance), the sugar consumption or the sugar consumption rate (the higher the sugar consumption or the sugar consumption rate, the better the performance), the yield as measured by ethanol (e.g., the ethanol production or the ethanol production rate (the higher the ethanol production or the ethanol production rate, the better the performance)) or gas (CO₂, e.g., the gas production or the gas production rate (the higher the gas production or the gas production rate, the better the performance)) production, the yeast biomass accumulation or the yeast biomass accumulation rate (an appropriate yeast biomass accumulation or yeast biomass accumulation rate, allowing propagation and fermentation at the same time limiting glycerol production) and/or tolerance to toxic environmental conditions (e.g., tolerance towards toxic compounds, towards elevated temperature, towards acidic or basic pH (the higher the tolerance, the better the performances)).

In some additional embodiments, the performance of methods of making a fermented product is not substantially altered when compared to the fermentation performance of a method conducted with a corresponding yeast host cell lacking the genetic modification. As used in the context of the present disclosure, the fermentation performance is not considered to be substantially altered (and in some embodiments, it may be increased) when compared to the fermentation performances of a method conducted with a corresponding yeast host cell lacking the genetic modification. As used in the context of the present disclosure, the term “is not substantially altered” refers to the fact that the fermentation performance can be reduced by at most 1 , 2, 5, 10, 15, 20 or 25% when compared to the fermentation performance of a method conducting with a corresponding host cell lacking the genetic modification. The performance during fermentation can be measured by determining the fermentation rate (the higher the fermentation rate, the better the performance), the sugar consumption or the sugar consumption rate (the higher the sugar consumption or the sugar consumption rate, the better the performance), the yield as measured by ethanol (e.g., the ethanol production or the ethanol production rate (the higher the ethanol production or the ethanol production rate, the better the performance)) or gas (CO₂, e.g., the gas production or the gas production rate (the higher the gas production or the gas production rate, the better the performance)) production, the yeast biomass accumulation or the yeast biomass accumulation rate (an appropriate yeast biomass accumulation or yeast biomass accumulation rate, allowing propagation and fermentation at the same time limiting glycerol production) and/or tolerance to toxic environmental conditions ( e.g ., tolerance towards toxic compounds, towards elevated temperature, towards acidic or basic pH (the higher the tolerance, the better the performances)).

In some additional embodiments, the performance of methods of making a fermented product is not substantially altered when compared to the fermentation performance of a method conducted with the recombinant host cell but in the absence of the pro-cytotoxic agent. As used in the context of the present disclosure, the fermentation performance is not considered to be substantially altered (and in some embodiments, it may be increased) when compared to the fermentation performances of a method conducted with a recombinant yeast host cell but in the absence of the pro-cytotoxic agent. As used in the context of the present disclosure, the term“is not substantially altered” refers to the fact that the fermentation performance can be reduced by at most 1 , 2, 5, 10, 15, 20 or 25% when compared to the fermentation performance of a method conducting with a recombinant host cell in the absence of the pro-cytotoxic agent. The performance during fermentation can be measured by determining the fermentation rate (the higher the fermentation rate, the better the performance), the sugar consumption or the sugar consumption rate (the higher the sugar consumption or the sugar consumption rate, the better the performance), the yield as measured by ethanol (e.g., the ethanol production or the ethanol production rate (the higher the ethanol production or the ethanol production rate, the better the performance)) or gas (CO₂, e.g., the gas production or the gas production rate (the higher the gas production or the gas production rate, the better the performance)) production, the yeast biomass accumulation or the yeast biomass accumulation rate (an appropriate yeast biomass accumulation or yeast biomass accumulation rate, allowing propagation and fermentation at the same time limiting glycerol production) and/or tolerance to toxic environmental conditions (e.g., tolerance towards toxic compounds, towards elevated temperature, towards acidic or basic pH (the higher the tolerance, the better the performances)).

The biomass that can be fermented to make the fermented product with the recombinant yeast host cell includes any type of biomass known in the art and described herein. For example, the biomass can include, but is not limited to, starch, sugar and lignocellulosic materials. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, molasses or sugar cane juice. The terms “lignocellulosic material”, “lignocellulosic substrate” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and/or agricultural residues, forestry residues and/or forestry wastes, paper-production sludge and/or waste paper sludge, waste -water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants and sugar-processing residues. The terms“hemicellulosics”, “hemicellulosic portions” and “hemicellulosic fractions” mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan and galactoglucomannan), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan) and proteoglycans (e.g., arabinogalactan-protein, extensin, and pro line -rich proteins).

In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, com cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.

Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxy methyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (A vice I), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose may be in a crystalline or noncrystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, molasses, sugarcane, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof. Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making, and is typically removed from process wastewater in a primary clarifier. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol. Processes provided by the present invention are widely applicable. Moreover, the saccharification and/or fermentation products may be used to produce ethanol or higher value added chemicals, such as organic acids, aromatics, esters, acetone and polymer intermediates.

In some embodiments, the present disclosure provides method for hydrolyzing a substrate comprising the biomass as described above, for example a substrate comprising molasses, sugar cane or a derivative therefrom (which can be referred to as a“must"), by contacting the substrate with a recombinant microbial host cell described herein. In some embodiments, the present disclosure provides a method for hydrolyzing a substrate, for example substrate comprising molasses, sugar cane or a derivative therefrom, by contacting the substrate with a co-culture comprising the recombinant microbial host cells described and another microorganism, such as, for example, a non-genetically-modified microorganism. In some embodiments, the method can also comprise including a purified enzyme to allow or facilitate the hydrolysis of the substrate or of an intermediary product made by the recombinant microbial host cell of the present disclosure.

The production of ethanol can be performed, for example, at temperatures of at least about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33° , about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C. In some embodiments, the production of ethanol from cellulose can be performed, for example, at temperatures above about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 43°C, or about 44°C, or about 45°C, or about 50°C. In some embodiments, the recombinant microbial host cell can produce ethanol from cellulose at temperatures from about 30°C to 60°C, about 30°C to 55°C, about 30°C to 50°C, about 40°C to 60°C, about 40°C to 55°C or about 40°C to 50°C.

In some embodiments, the production of ethanol (or other products and co-products) can further be performed according to the "Brazil process." Under the "Brazil process," non-sterilized cane juice and/or molasses is fermented at a high inoculum to achieve fast fermentations. During the fermentation process, the yeast is repeatedly recycled over the 200+ day crop season by centrifuging the cells and washing them in sulphuric acid to decrease contamination and break up flocculation of cells. Industrial strains isolated from ethanol fermentations in Brazil have been shown to have characteristics that allow them to survive the acid washing and fermentation conditions better than typical lab yeast or other industrial yeast isolates. PE-2, is a wild isolate from cane ethanol fermentation. PE-2 and other industrial strains produce an average of 4.5 g/L glycerol. In some embodiments, the PE-2 strain, or a modified version thereof, is used as the host organism. In certain embodiments, ethanol is produced through the fermentation of a recombinant yeast host cell according to the Brazil process. In some embodiments, the recombinant yeast host cell is used to ferment a carbohydrate source wherein the yeasts are reused after one or more fermentations (e.g., cycles), and wherein the yeasts are washed with an acid (e.g., acid washed) following each fermentation. In some embodiments, the acid has a pH of between 2.0 and 2.2. In certain embodiments, the acid is sulphuric acid. In some additional embodiments, the acid washing cycle can be repeated more than once, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more acid washing cycles can be performed. In some embodiments, methods of producing ethanol can comprise contacting the substrate with a recombinant yeast host cell or co-culture as described herein and additionally contacting the substrate with externally produced enzymes which can be provided in a purified form. Exemplary externally produced enzymes include, but are not limited to starch degrading enzymes, dextran degrading enzymes, phytase, protease, cellulases and/or xylose isomerase. Specific externally produced (and optionally purified) enzymes include, but are not limited to, trehalases, glucoamylases, alpha-amylases, alpha- glucosidases, glucanases (endo/exo), pullulanases, phytases and/or proteases.

The process of the present disclosure can include a step of inoculating the recombinant yeast host cell in a medium comprising a carbohydrate source (and optionally a pro-cytotoxic agent). An inoculated medium can be referred to as a must. In some embodiment, the must can be from obtained from a sugar cane. After the recombinant yeast host cell has been inoculated in the first medium, it is submitted to a fermentation step to generate a fermentation product. The process can also include, once the fermentation has been completed or has been stopped, dissociating the solid portion (recombinant yeast host cells and other associated the solids) from the liquid portion of the fermented medium. This dissociation step can be achieved, for example, using centrifugation and/or filtration. The solid and/or the liquid portion can be further treated, for example, to purify (at least in part), the fermentation production from the solid and/or the liquid portion of the fermentation product. The process can include further treating the solid portion comprising the recombinant yeast host cell with one or more acid wash. In some embodiments, the pro-cytotoxic agent can be further added to the recombinant yeast host cells before, during or shortly after the acid wash step. In processes including an acid wash step, the acid washed recombinant yeast host cells can be added to a further medium (which can optionally include the pro-cytotoxic agent) and submitted to a further fermentation cycle.

In embodiments in which the recombinant yeast host cells are used to produce ethanol according to the Brazilian process, the process for limiting microbial contamination can be adapted to be performed in coordination. For example, the process can be conducted prior to or after the acid washing step. In yet another example, the process can be conducted at each fermentation cycle, once every two fermentation cycles, once every three fermentation cycles, once every four fermentation cycles, once every five fermentation cycles or once every six fermentation cycles, etc. In still another example, the process can be each on a weekly or monthly basis. All the steps of the process for limiting microbial contamination may be required to be completed, however, in some instances, it is contemplated that only the monitoring will be necessary (either because the first threshold is not exceeded or the second threshold is not reached).

In some embodiments, the methods comprise producing ethanol at a particular rate. For example, in some embodiments, ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, at least about 1 g per hour per liter, at least about 1.5 g per hour per liter, at least about 2 g per hour per liter, at least about 2.5 g per hour per liter, at least about 3 g per hour per liter, at least about 3.5 g per hour per liter, at least about 4 g per hour per liter, at least about 4.5 g per hour per liter, at least about 5 g per hour per liter, at least about 5.5 g per hour per liter, at least about 6 g per hour per liter, at least about 6.5 g per hour per liter, at least about 7 g per hour per liter, at least about 7.5 g per hour per liter, at least about 8 g per hour per liter, at least about 8.5 g per hour per liter, at least about 9 g per hour per liter, at least about 9.5 g per hour per liter, at least about 10 g per hour per liter, at least about 10.5 g per hour per liter, at least about 11 g per hour per liter, at least about 11 .5 g per hour per liter, at least about 12 g per hour per liter, at least about 12.5 g per hour per liter, at least about 13 g per hour per liter, at least about 13.5 g per hour per liter, at least about 14 g per hour per liter, at least about 14.5 g per hour per liter or at least about 15 g per hour per liter. In some embodiments, the ethanol can be produced in the absence of any externally added cellulases.

Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - STRAIN DESCRIPTION

The following strains have been used in the Examples:

EXAMPLE II -ASSAY FOR MONITORING CONTAMINATION

Strains M 10682 and PE-2 were individually grown on YPD, mixed at various ratios (as indicated in the brief description of the drawings section for Figure 2A) and plated on YPD agar plates lacking and having 500 mg/mL 5-FC. All yeast strains were able to grow on the YPD agar plates in the absence of 5-FC (Figure 2A, top panels). Only strain M 10682 was able to grow on the YPD agar plates containing 5-FC (Figure 2A, bottom panels).

Individual M 10682 colonies were isolated and inoculated into 600 mL of YPD in a 96-well plate. Three microliters were spotted to a YPD agar plates and a YPD + 500 mg/mL 5-FC agar plates. Plates were incubated at 35°C overnight. Controls are shown in the box. Strain PE-2 showed normal growth on YPD agar but was unable to grow on plates containing 5-FC (Figure 2B). Strain M 10682 was able to grow on both the YPD and the 5-FC agar plates (Figure 2B). The other 40 colonies shown on Figure 2B(ii) are M 10682 isolates that have been grown for 65 generations prior to spotting and show that the ability to grow on this 5-FC media is a stable phenotype and could consistently be used to detect M10682.

It was also shown that strain M 10682 can also be specifically detected in liquid media containing 5-FC. Samples from scaled down industrial fermentations on PE-2 and M 10682 were inoculated directly into a YPD media or a YPD supplemented with 500 mg/mL 5-FC (YPD+5- FC). Both strains were able to grow in the base media as detected by the yeast cell pellet in the bottom of the tube as well as the color change (purple to yellow) of the liquid that is indicative of yeast growth and a decrease in the pH of the media (Figure 2C(i)). In YPD+5FC, PE-2 was unable to grow while M10682 growth was detectable by the yeast cell pellet and color change of the media (purple to yellow) (Figure 2C(ii)).

EXAMPLE III - LIMITING CONTAMINATION DURING FERMENTATION WITH A FCY1

DELETION

Strain M10682 was mixed with 1 %, 5% or 10% of strain PE-2. Each of the M10682+PE-2 yeast mixed populations completed four rounds of fermentation and yeast cell recycle in the present of 0, 1 or 4 ppm 5-FC. At the end of each fermentation cycle the yeast were outgrown for seven generations, DNA was extracted from the total population and qPCR was used to determine the relative abundance of M10682 and PE-2. As shown on Figure 3, the addition of 5-FC to the medium during fermentation substantially reduced the contamination by strain PE-2. Even the 10% PE-2 contamination can be eliminated in 2 cycles of treatment with 4 ppm 5-FC or 4 cycles of treatment with 1 ppm 5-FC (Figure 3C).

EXAMPLE IV - LIMITING CONTAMINATION DURING FERMENTATION WITH A FCY1/FUR1

DELETION

A supplemental deletion has been introduced in the furl gene (e.g., strain M15980, see table 1 above). Fermentations were conducted in a commercial sourced must with 0 (dark grey) or 50 (light gray) ppm 5-FU. Strains were either grown alone or in mixes as indicated. Final ethanol concentrations were determined by HPLC As shown in Figure 4, in medium supplemented with 5-FU, the ethanol production of both PE-2 and M10682 were severely inhibited. However, the ethanol production of a strain comprising a deletion in both the fcy1 and the furl gene was maintained both in the presence and in the absence of 5-FU.

Strains M10682 and M15980 were each mixed with 10%, 50% or 75% of wild-type contaminating stain PE-2. Fermentations were conducted in a commercial sourced must with 0 (dark grey) or 10 (light gray) ppm 5-FC. Strains were either grown alone or in mixes as indicated. In the absence of 5-FC, strain M15980 performed similarly to strain M10682 when challenged with contaminating wild-type strain PE-2 (Figure 5A). However, in the presence of 5- FC, the strains performed differently: strain 10682 did not surive 5-FC treatment, while strain M15980 thrived after 5-FC treatment (Figure 5B). Without wishing to be bound to theory, it is stipulated that, at high levels of contamination with the PE-2 strain (e.g., above 10%), the PE-2 strain converted the 5-FC into the cytotoxic 5-FU which was harmful to both the PE-2 and the M10682 strains. However, at the same high levels of contamination with the PE-2 strain, the 5- FC converted to 5-FU was only harmful to the PE-2 strain and not the M15980 strain. The population testing determined that that 10 ppm 5-FC killed all yeast in the M10682+PE-2 fermentation mixtures (no colonies formed on permissive of selective plate) but that the PE-2 cultures that were mixed with 88%, 47% or 24% M15980 became 100%, 98% and 89% of strain M15980 at the end of 5-FC treatment.

As shown in Figure 6, it is expected that a deletion of the FUR1 gene alone (DFur1) is expected to give similar resistance and performance as a double deletion of FCY1 and FUR1 (DFCY1 DFur1).

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:

1. A process for preventing or limiting a microbial contamination caused by contaminating microorganisms during a continuous culture of a recombinant yeast host cell, the process comprising culturing the recombinant yeast host cell in the presence of a pro-cytotoxic agent, wherein the recombinant yeast host cell has a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of the pro- cytotoxic agent into a cytotoxic agent.

2. The process of claim 1 , wherein the genetic modification comprises disrupting the open reading frame of the noxious gene.

3. The process of claim 1 or 2, wherein the genetic modification comprises increasing the expression of a gene encoding an inhibitor of expression of the noxious gene.

4. The process of any one of claims 1 to 3 comprising:

(a) culturing the recombinant yeast host cell in a first medium lacking the pro-cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first medium comprises a first total microbial population and the first threshold of contaminating microorganisms is at least above about 0.1 % of the first total microbial population;

(b) when the first threshold of contaminating microorganisms is reached in the first medium, culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second medium has a second total microbial population and the second threshold < 0.1 % of the second total microbial population; and

(c) if or when the second threshold of contaminating microorganisms is reached in the second medium, culturing the recombinant yeast host cell in the first medium lacking the pro-cytotoxic agent.

5. The process of claim 4 comprising culturing the recombinant yeast host cell in the first medium when the second threshold is reached.

6. The process of any one of claims 1 to 3 comprising:

(a) culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second medium comprises a second total microbial population and the second threshold of contaminating microorganisms £ 0.1 % of the second total microbial population;

(b) when the second threshold of contaminating microorganisms is reached in the second medium, culturing the recombinant yeast host cell in a first medium lacking the pro- cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first medium has a first total microbial population and the first threshold is above about 0.1 % of the first total microbial population; and

(c) if or when the first threshold of contaminating microorganisms is exceeded in the first medium, culturing the recombinant yeast host cell in the second medium lacking the pro- cytotoxic agent.

7. The process of claim 6 comprising culturing the recombinant yeast host cell in the second medium when the first threshold is exceeded.

8. The process of any one of claims 4 to 7, wherein steps (a) and (b) are repeated after step (c).

9. The process of any one of claims 4 to 8 comprising monitoring, in the first medium, the percentage of contaminating microorganisms with respect to the first total microbial population.

10. The process of any one of claims 4 to 8 comprising monitoring, in the second medium, the percentage of contaminating microorganisms with respect to the second total microbial population.

11. The process of claim 9 or 10, wherein the monitoring is done at least once a month.

12. The process of claim 9 or 10, wherein the monitoring is done at least once a week.

13. The process of any one of claims 9 to 12, wherein the monitoring comprises assessing the number of colony-forming units of the contaminating microorganisms to monitor the percentage of contaminating microorganisms.

14. The process of any one of claims 4 to 13 comprising adding the pro-cytotoxic agent to the first medium to obtain the second medium.

15. The process of any one of claims 4 to 14 comprising refraining from adding the pro- cytotoxic agent to the second medium to obtain the first medium.

16. The process of claim 1 comprising culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent during the continuous culture.

17. The process of any one of claims 1 to 16, wherein the noxious gene is at least one of FCY1 , FUR1 , URA3, LYS2, LEU2, TRP1 , HIS3, MET15 or ADE2.

18. The process of claim 17, wherein the noxious gene comprises FCY1.

19. The process of claim 18, wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC).

20. The process of claim 17, wherein the noxious gene is FUR1.

21. The process of claim 17, wherein the noxious gene comprises FCY1 and FUR1 .

22. The process of claim 20 or 21 , wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU).

23. The process of claim 18 to 22, wherein the concentration of 5-FC or 5-FU is between about 0.1 and about 500 ppm in the second medium.

24. The process of any one of claims 1 to 23 comprising contacting the pro-cytotoxic agent with the recombinant yeast host cell at least once, twice or thrice a year.

25. The process of any one of claims 1 to 24, wherein the contaminating microorganisms comprise yeasts.

26. The process of any one of claims 1 to 25, wherein the recombinant yeast host cell is from Saccharomyces sp.

27. The process of claim 26, wherein the recombinant yeast host cell is from Saccharomyces cerevisiae.

28. A process for making a fermentation product from a first and/or a second fermentation medium comprising a carbohydrate, the process comprising culturing a recombinant yeast host cell in the presence of a pro-cytotoxic agent under conditions so as to allow making the fermentation product, wherein the recombinant yeast host cell has a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of the pro-cytotoxic agent into a cytotoxic agent.

29. The process of claim 28, wherein the genetic modification comprises disrupting the open reading frame of the noxious gene.

30. The process of claim 28 or 29, wherein the genetic modification comprises increasing the expression of a gene encoding an inhibitor of expression of the noxious gene.

31. The process of any one of claims 28 to 30 comprising:

(a) contacting the recombinant host cell with the first fermentation medium comprising a carbohydrate and lacking the pro-cytotoxic agent under conditions to promote the production of the fermentation product and until a first threshold of contaminating microorganisms is exceeded, wherein the first fermentation medium comprises a first total microbial population and the first threshold of contaminating microorganisms is above about 0.1 % of the first total microbial population;

(b) when the first threshold of contaminating microorganisms is exceeded in the first fermentation medium, culturing the recombinant yeast host cell in the second fermentation medium having the carbohydrate and the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second fermentation medium has a second total microbial population and the second threshold £ 0.1 % of the second total microbial population; and

(a) if or when the second threshold of contaminating microorganisms is reached in the second fermentation medium, culturing the recombinant yeast host cell in the first fermentation medium.

32. The process of claim 31 comprising culturing the recombinant yeast host cell in the first fermentation medium when the second threshold is reached.

33. The process of any one of claims 28 to 30, wherein the process comprises:

(a) culturing the recombinant yeast host cell in a second fermentation medium having a carbohydrate and the pro-cytotoxic agent until a second threshold of contaminating microorganisms is reached, wherein the second fermentation medium comprises a second total microbial population and the second threshold of contaminating microorganisms £ 0.1 % of the second total microbial population;

(b) when the second threshold of contaminating microorganisms is reached in the second fermentation medium, culturing the recombinant yeast host cell in a first fermentation medium comprising the carbohydrate and lacking the pro-cytotoxic agent until a first threshold of contaminating microorganisms is exceeded, wherein the first fermentation medium has a first total microbial population and the first threshold is between about 1 - 10% of the first total microbial population; and

(c) if or when the first threshold of contaminating microorganisms is exceeded in the first fermentation medium, culturing the recombinant yeast host cell in the second fermentation medium.

34. The process of claim 33 comprising culturing the recombinant yeast host cell in the second fermentation medium when the first threshold is exceeded.

35. The process of any one of claims 31 to 34 comprising repeating steps (a) and (b) after step (c).

36. The process of any one of claims 28 to 32 comprising monitoring, in the first fermentation medium, the percentage of contaminating microorganisms with respect to the first total microbial population.

37. The process of any one of claims 28 to 33 comprising monitoring, in the second fermentation medium, the percentage of contaminating microorganisms with respect to the second total microbial population.

38. The process of claim 36 or 37, wherein the monitoring is done at least once a month.

39. The process of claim 36 or 37, wherein the monitoring is done at least once a week.

40. The process of any one of claims 36 to 39, wherein the monitoring comprises assessing the number of colony-forming units of the contaminating microorganisms to monitor the percentage of contaminating microorganisms.

41. The process of any one of claims 31 to 40 comprising adding the pro-cytotoxic agent to the first fermentation medium to obtain the second fermentation medium.

42. The process of any one of claims 31 to 40 comprising refraining from adding the pro- cytotoxic agent to the second fermentation medium to obtain the first fermentation medium.

43. The process of claim 31 comprising culturing the recombinant yeast host cell in a second medium having the pro-cytotoxic agent during the continuous culture.

44. The process of any one of claims 28 to 43, wherein the noxious gene is at least one of

FCY1 , FUR1 , URA3, LYS2, LEU2, TRP1 , HIS3, MET15 or ADE2.

45. The process of claim 44, wherein the noxious gene comprises FCY1.

46. The process of claim 45, wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC).

47. The process of claim 44, wherein the noxious gene is FUR1.

48. The process of claim 44, wherein the noxious gene comprises FCY1 and FUR1 .

49. The process of claim 47 or 48, wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU).

50. The process of any one of claims 31 to 49, wherein the second fermentation medium comprises between about 0.1 and about 500 ppm of 5-FC or 5-FU.

51. The process of any one of claims 31 to 50 comprising contacting the pro-cytotoxic agent with the recombinant yeast host cell at least once, twice or thrice a year.

52. The process of any one of claims 31 to 50, wherein the contaminating microorganisms comprise yeasts.

53. The process of any one of claims 31 to 52, wherein the carbohydrate is a sugarcane juice, a sugarcane derivative, com, a corn derivative, molasses and/or a molasses derivative.

54. The process of any one of claims 31 to 53, wherein the fermentation product is ethanol, isopropanol, n-propanol, 1 -butanol, methanol, acetone, 1 , 2 propanediol or an heterologous polypeptide.

55. The process of any one of claims 31 to 54 further comprising contacting the procytotoxic agent with the recombinant yeast host cell prior to culturing the recombinant yeast host cell.

56. The process of any one of claims 31 to 55 further comprising at least one fermentation cycle comprising acid washing the recombinant yeast host cell present in the first and/or second fermentation medium to obtain an acid washed recombinant microbial host cell and contacting the acid washed recombinant yeast host cell with the first and/or the second fermentation medium to promote the production of the fermentation product.

57. The process of claim 56, further comprising at least two or more fermentation cycles.

58. The process of claim 56 or 57 comprising performing steps (a), (b) and (c) prior to acid washing.

59. The process of any one of claims 56 to 58 comprising performing steps (a), (b) and (c) at least once in each fermentation cycle.

60. The process of any one of claims 31 to 59, wherein the genetic modification of the noxious gene does not substantially alter the fermentation performance of the recombinant yeast host cell when compared to the fermentation performance of a corresponding yeast host cell lacking the genetic modification.

61. The process of any one of claims 31 to 60, wherein the recombinant yeast host cell is from Saccharomyces sp.

62. The process of claim 61 , wherein the recombinant yeast host cell is from Saccharomyces cerevisiae.

63. A method of determining the presence of contaminating microorganisms in a specimen having a total microbial population and comprising a recombinant yeast host cell, the recombinant yeast host cell having a genetic modification for reducing the expression of a noxious gene and wherein the genetic modification impedes the conversion of a pro-cytotoxic agent into a cytotoxic agent, the method comprising:

(a) culturing a first sample of the specimen in a selective medium comprising the pro- cytototoxic agent to determine the presence of recombinant yeast host cells in the specimen;

(b) culturing a second sample of the specimen in a permissive medium lacking the pro- cytotoxic agent to determine the total microbial population of the specimen; and

(c) determining the presence of contaminating microorganisms in the specimen based on the determination made in steps (a) and (b).

64. The method of claim 63, wherein the noxious gene is FCY1.

65. The method of claim 64, wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC).

66. The method of claim 63, wherein the noxious gene is FUR1.

67. The method of claim 66, wherein the noxious gene comprises FCY1 and FUR1.

68. The method of claim 66 or 67, wherein the pro-cytotoxic agent is 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU).