Fermentation process

Abstract

The invention fermentation processes for recombinantly producing a target peptide. The present disclosure also relates to a yeast cell comprising a deletion or modification in an endogenous gene AMN1, encoding Amn1 protein, and a polynucleotide vector encoding a target peptide, and to uses of said yeast cell.

Description

[0001] FERMENTATION PROCESS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to fermentation processes for recombinantly producing a target peptide. The present disclosure also relates to a yeast cell comprising a deletion or modification in an endogenous gene encoding Amn1 protein and a polynucleotide vector encoding a target peptide. The present disclosure further relates to uses of said yeast cell.

[0004] BACKGROUND

[0005] Microbial fermentations are widely used for the production of various industrial and pharmaceutical products, including antibiotics, organic acids, amino acids, peptides, proteins, vitamins, and polymers. Increased productivity and recovery of more highly purified product are major areas of development to increase profitability. There are several factors that can affect the efficiency and productivity of fermentation processes. One of the major challenges in fermentation processes is the control of the viscosity of the fermentation broth.

[0006] The high viscosity of fermentation broth can pose significant challenges for the reproducible and / or efficient production of high-quality target peptides. For instance, as the viscosity increases, maintaining proper mixing becomes more challenging, which can negatively impact cell growth, and decrease product formation rates. Poor mixing and aeration can result in decreased mass transfer of nutrients, oxygen, and waste products, which can negatively impact cell growth and product yields. In addition, high viscosity can lead to increased energy consumption during agitation, which can have a negative impact on the overall efficiency of the fermentation process.

[0007] In aerobic fermentations, a higher agitation speed or rate is required to maintain the same oxygen transfer rate as the viscosity increases. However, commercial fermentation plants are often operated close to the maximum agitation speed, which means that increased viscosity can decrease the oxygen transfer rate and impact growth and productivity.

[0008] To meet the growing demand for these products, there is a need to increase the productivity and yield of fermentation processes.

[0009] Hence, there is a need for improved methods to control the viscosity of fermentation broth in a safe, efficient, and cost-effective manner.

[0010] SUMMARY

[0011] It is an object to improve target peptide production in recombinant host cells and / or to increase the recovery rate of a target peptide. Another object is to provide an improved variant yeast host cell comprising a deletion or modification or disruption in an endogenous gene, that allow for a decrease in viscosity of the respective fermentation liquid compared to the viscosity of the fermentation liquid when using the parental host cell. Yet another object is the use of a variant yeast host cell comprising a deletion or a disruption or a modification in an endogenous gene encoding Amn1 protein for the recombinant production of a target peptide.

[0012] Also, or alternatively, it is an object of the present disclosure to address one or more of the aforementioned problem(s) or of the problems mentioned further below. The disclosure may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.

[0013] The one or more object(s) is solved by the subject matter as claimed.

[0014] Even though the primary parameter affected by the present disclosure is the viscosity of the fermentation broth, the skilled person using the present disclosure may observe a number of benefits such as, improved liquid mixing, reduced spatial variation of important fermentation parameters, improved oxygen uptake, improved carbon dioxide removal, facilitating the efficient dissipation of metabolic and mechanically-induced heat and thus lowering cooling requirements, higher productivity and improved yield. The disclosure may also allow for an improved oxygen transfer rate (see relationship (1)), less substrate and oxygen granted, and shorter mixing times. See, e.g., Buckland, B.C., Gbewonyo, K., Jain, D., Glazomitsky, K., Hunt, G. and Drew, S.W. (1988). Oxygen transfer efficiency of hydrofoil impellers in both SOOL and 1900L fermenters. In Proceedings of the second international conference on bioreactor fluid dynamics, pp. 1-16 R. King (Ed.). London: Elsevier.

[0015] In a first aspect, the present disclosure relates to a fermentation process for expressing a target peptide. The fermentation process comprises the steps of preparing a fermentation medium, inoculating the fermentation medium with a yeast cell comprising a DNA sequence encoding said target peptide, adjusting the pH of the inoculated fermentation medium to a desired pH setpoint. The pH setpoint is in the range of 3.0-6. The pH is typically kept constant at said pH setpoint. The inoculated fermentation medium is cultivated at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth. The fermentation broth is then clarified to obtain a soluble fraction, optionally by centrifugation. The target peptide can be recovered by adjusting the pH of the supernatant to a pH value in the range of 7.5 to 12.0. The recovered target peptide may then be purified.

[0016] In a second aspect, the present disclosure relates to a method for producing a target peptide. The method for producing a target peptide comprises the steps of transforming a genetically modified yeast cell with a polynucleotide vector encoding a target peptide, wherein the yeast cell comprises an AMN1 gene and wherein the D368 allele of the AMN1 gene has been knocked out or has been modified to a D368\ / allele of the yeast cell has been deleted or disrupted or modified, and inoculating a fermentation medium with the transformed yeast cell. The inoculated fermentation medium can be adjusted to a desired pH setpoint, wherein the pH setpoint is above 5.5. The pH is then typically kept constant at said pH setpoint. The inoculated fermentation medium can be cultivated at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth to then harvest the fermentation broth. Afterwards the fermentation broth can be clarified to obtain a supernatant, optionally by centrifugation, and recover the target peptide. The recovered target peptide may be purified.

[0017] In a third aspect, the present disclosure relates to a variant yeast cell derived from a parental cell comprising an endogenous gene encoding Amn1 protein, wherein the variant yeast cell comprises a deletion or disruption or modification in an endogenous gene encoding Amn1 protein, and wherein the variant yeast cell further comprises a polynucleotide vector encoding a target peptide. The variant yeast cell is capable of expressing the target peptide in higher yields at a pH above 5.5, such as at a pH in a range of about 5.5-12, than at a pH below 5.5, such as at a pH in range of 5.4-1. In particular embodiments of the third aspect, the variant yeast cell is a Saccharomyces cerevisiae cell, wherein the cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant cell has been modified such that the D368 allele has been knocked out or has been modified to a D368V allele; and wherein the variant cell further comprises a polynucleotide vector encoding a target peptide. Conveniently, the target peptide may be an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0018] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0019] Z2is an optional polypeptide or amino acid residue;

[0020] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0021] X5is selected from the genetically encoded amino acids but S and I;

[0022] X6is absent or selected from the genetically encoded amino acids; and

[0023] Z1 is selected from the group consisting of K34R-GLP-1(7-37), K34R-GLP-1(9- 37), EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS, and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS. In a fourth aspect, the present disclosure relates to the use of a variant yeast cell comprising a deletion or modification in an endogenous gene encoding Amn1 protein for the recombinant production of a target peptide. Particular embodiments of the fourth aspect relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been deleted. Further or alternative particular embodiments of the fourth aspect relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP- 1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele.

[0024] Particular embodiments of the fourth aspect relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1-GIP polypeptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been deleted. Further or alternative particular embodiments of the fourth aspect relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1GIP polypeptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele.

[0025] BRIEF DESCRIPTION OF DRAWINGS

[0026] The embodiments of the disclosure, together with its advantages, may be best understood from the following description taken in conjunction with the accompanying figures.

[0027] Fig. 1 shows the relative target peptide concentration from a pH 5.3 cultivation of the yNN2071 strain expressing GLP-1 precursor ([Arg34]GLP- 1(9-37)) with N-terminal extension 1 as measured by a) the dilution method (•) and b) alkalic extraction method (O) to de-aggregate the target peptide.

[0028] Fig. 2A shows an example of a rheological characterisation of fermentation broth from yNN2071 cultivated at pH 5.3 expressing GLP-1 precursor ([Arg34]GLP- 1(9-37)) with N-terminal extension 1. The viscosity at different shear rates of the fermentation broth (♦), fermentation supernatant (O) and re-suspended washed pellet (•) was analysed. Fig. 2B shows the viscosity of the fermentation broth standing still on the lab bench at 20 °C before ( ) and after vortexing for 2 minutes (•)

[0029] Fig. 3 shows the correlation between the relative concentration of the aggregated target peptide (calculated by subtracting the relative concentration derived by the dilution method from the relative concentration derived by the alkalic method) and broth flow behaviour index of the yNN2071 strain cultivated at pH 5.3. The dotted line represents the best fit linear regression line. Fig. 4 shows the correlation between fermentation broth pH, relative target peptide concentration (•) viscosity at 2000 s'1(□) and rheological behaviour (i.e. flow behaviour index n (♦) and flow consistency index k (O)) for yNN2071 cultivation.

[0030] Fig. 5 shows the relative target peptide concentration in the supernatant of yNN2071 (using a cultivation pH of 5.3) and of two strains from a different host cell lineage, yNN2101 and yNN2104 (using a cultivation pH of 6.2) with and without alkalic treatment of the supernatant.

[0031] Fig. 6A shows the target peptide concentration from a pH 6.2 cultivation of yNN2104 as measured by the dilution method (•) and alkalic extraction method (O) to de-aggregate the target peptide. Fig. 6B shows the dissolved oxygen tension (DO2) (— ), oxygen uptake rate (OUR) ( — ) and viscosity (O) against cultivation time using yNN2104.

[0032] Fig. 7A shows an example of the rheological characterisation of fermentation broth from strain yNN2104 expressing a target peptide (GLP-1 precursor ([Arg34]GLP-1 (9-37)) with N-terminal extension 1) and Fig. 7B shows an example of the rheological characterisation of fermentation broth from a non-target peptide expressing strain (yNN1953) of the same host cell lineage. The viscosity at different shear rates of the fermentation broth (♦), fermentation supernatant (O), and resuspended washed pellet (•) was analysed.

[0033] Fig. 8 shows the effect of cell flocculation by Ca2+on the rheological properties of yeast cell suspensions of strain yNN2201 expressing GLP1-GIP fusion precursor with extension 2. Fermentation broth samples were a) double-washed and resuspended pellet in de-ionized water (O), b) double-washed and resuspended pellet in a 0.02M EDTA solution ( ), c) double-washed and resuspended pellet in a 25% (w / v) CaCI2solution (•).

[0034] Fig. 9. The cultivation broth rheological behaviour and relative target peptide concentration determined by the dilution and alkalic extraction method of three target peptide producing strains (yNN2548, yNN2101, yNN2104) and one non-producing (dummy) strain (yNN1953) before and after the addition of Ca2+(in the form of CaCI2). Fig. 9A shows the viscosity at shear rate 2000s-1, Fig. 9B shows the flow consistency index k (mNsnrrr2) and Fig. 9C shows the flow behaviour index n against cultivation time for different strains. The bold dotted line @ ~409h shows the addition of calcium chloride (0.05M in continuous substrate, DOS2). Fig. 9D shows the average flow behaviour index n before and after the addition of Ca2+. The average flow behaviour index values were determined by integrating the flow behaviour index over the respective time intervals using the composite trapezoidal rule and then dividing by the duration of those intervals. Figs. 9E-G shows the target peptide extracted by dilution and alkalic treatment before and after the addition of Ca2+. Fig. 10A shows the viscosity at a shear rate of 2000 S'1against cultivation time. Fig. 10B shows the flow behaviour index n against cultivation time of the parental strain (yNN2104, WT) (•) and derivative strains with deletion of FLO8 (yNN2912) (♦) and FLO11 genes (yNN2915) (O)-

[0035] Fig. HA shows the viscosity at 250 S’1, Fig. H B shows the Flow behaviour index (n), Fig. HC shows the dissolved oxygen tension (%), Fig. 11 D shows the oxygen uptake rate (mlWh), of yNN2104 (AMN1 WT) (•) versus yNN2993 (AAMNT) ( ) against cultivation time. Fig. 11E shows a microscopic image of cells from yNN2104 (AMN1 WT) and yNN2993 (AAMN1).

[0036] Fig. 12A shows dissolved oxygen tension (%). Fig. 12B shows the oxygen uptake rate. Fig. 12C shows the relative dry cell weight. Fig. 12D shows the viscosity at 2000 S’1, Fig. 12E shows the flow behaviour index n, and Fig. 12F shows the relative target peptide concentration of the yNN2104 (AMN1 WT) (•), yNN2993 (AAMN1) (x) and yNN3073 (AMN1D368V) ( ) against cultivation time.

[0037] Fig. 13 shows the viscosity (cP) at shear rate 250s-1(O), flow behaviour index n (♦), aggregation index (•) which is the concentration as determined by the alkalic extraction method divided by the dilution extraction method and cultivation pH ( — ) of the yNN2993 (AAMNT) against cultivation time. Examples of the viscosity at different shear rates of the fermentation broth ( □ ), fermentation supernatant (O), and re-suspended washed pellet (•) before and after the change of cultivation pH from 6.4 to 5.3 are also shown.

[0038] Fig. 14A shows the viscosity at 2000 s-1and Fig. 14B shows the flow consistency index against cultivation time for haploid strains derived from the model strain EM93: yNN0381 (AMN1 WT) (•), yNN3497 (AAMN1) (♦) and yNN3521 (A / W / V7D3SS' / J (x). In the case of AAMN1 and AMN1D368Vthe results are represented as the average of two fermentations and the error bars represent their standard deviation. The shear rate ranges used for the calculation of flow consistency index are 150 to 4000 s’1for the high viscosity samples and 500 to 4000 s_1for the low viscosity samples.

[0039] Fig. 15A shows the viscosity at shear rate 250 s_1and Fig. 15B shows the flow behavioural index against cultivation time for yNN2071 cultivated at pH 5.3 (O), and two strains of different host cell lineage: yNN2104 with AMN1 WT cultivated at pH 6.2 (•) and yNN2993 with AMN1 deletion cultivated at pH 6.5 (Q).

[0040] Fig. 16 shows the following cultivation variables of strains yNN7092 (AAMN1) expressing GLP-1 precursor with extension 3 (ext3-GLP1) (o ) , yNN7093 (AAMN1) expressing GLP-1 precursor with extension 4 (ext4-GLP1) (O ) , yNN7095 (AMN1 WT) expressing GLP-1 precursor with extension 3 (ext3-GLP1) (• ) , yNN7096 (AMN1 WT) expressing GLP-1 precursor with extension 4 (ext4-GLP1) ( ) : A: the cultivation pH during the course of the cultivations of the strains. B: the relative target peptide concentrations. C: the relative aggregated target peptide concentrations. Di:the viscosity of the cultivation broth at the shear rate of 2000 S’1. D zoom in of the period where the cultivation pH was set at 6.5. Ei:the flow behaviour index. Em zoom in of the period where the cultivation pH was set at 6.5.

[0041] Fig. 17 A. shows the viscosity at different shear rates of the supernatant coming from the broth of strain yNN7093 (AAMN1) expressing GLP-1 precursor with extension 4 (target peptide) adjusted to pH 5.26 (• ), 5.92 (■), 6.64 (A), 7.36 (♦), 8.18 (+) and 9.89 ( X) before clarification. Fig. 17B. summarizes the results from A showing the flow behaviour (• ) and consistency (■) indices, the viscosity at shear rate of 2000 S’1(A) and the relative aggregated target peptide (+) against clarification pH.

[0042] Fig. 18 shows i. the dissolved oxygen tension, ii. the oxygen uptake rate, iii. respiratory quotient and iv. relative dry cell weight of cultivated strains (A) yNN8083 (AMN1 WT) expressing the GLP1-GIP precursor 1 with extension 5 , (— ) yNN8079 ( AMN1) expressing the GLP1-GIP precursor 1 with extension 5 (— ) ; (B) yNN8084 (AMN1 WT) expressing the GLP1-GIP precursor 1 with extension 6 , (•— ) , yNN8080 (AAMN1) expressing the GLP1-GIP precursor 1 with extension 6 (— ) ; (C) yNN8085 (AMN1 WT) expressing GLP1-GIP precursor 1 without extension , (— ) , yNN8081 (AAMN1) expressing the GLP1-GIP precursor 1 without extension (— ) ; (D) yNN8086 (AMN1 WT) expressing the GLP1-GIP precursor 2 without extension , (— ) , yNN8082 (AAMN1) expressing the GLP1-GIP precursor 2 without extension ( — ) against cultivation time.

[0043] Fig. 19 shows i. the broth viscosity at shear rate of 2000 S’1, ii. the relative target peptide concentration and iii. the relative aggregated target peptide concentration of the cultivated strains (A) yNN8083 (AMN1 WT) expressing the GLP1-GIP precursor 1 with extension 5 , (• ) , yNN8079 (AAMN1) expressing the GLP1-GIP precursor 1 with extension 5 (o ) ; (B) yNN8084 (AMN1 WT) expressing the GLP1-GIP precursor 1 with extension 6 , (■ ), yNN8080 (AAMN1) expressing the GLP1-GIP precursor 1 with extension 6 (□ ) ; (C) yNN8085 (AMN1 WT) expressing the GLP1-GIP precursor 1 without extension (□ ), yNN8081 (AAMN1) expressing the GLP1-GIP precursor 1 without extension (□ ) ; (D) yNN8086 (AMN1 WT) expressing the GLP1-GIP precursor 2 without extension (A) , yNN8082 (AAMN1) expressing the GLP1-GIP precursor 2 without extension (□ ) against cultivation time.

[0044] Fig. 20. Bar charts of the average viscosity at shear rate of 2000 S'1(determined by integrating the viscosity values over the respective time intervals using the composite trapezoidal rule (trapz function from NumPy v1.26) and then dividing it by the duration of those intervals) of broth from cultivation of (■ ) AMN1 WT strains expressing different target peptides and (■ ) AAMN1 strains expressing different target peptides before and after the 320h timepoint where the cell- derived viscosity increase occurs for the AMN1 WT strains. The error bars represent the standard deviation of the viscosity values within the specified time intervals. This indicates the natural variability of the data over these periods, rather than measurement uncertainty. Statistical analysis was performed using the Welch's t-test for the comparison between the AMN1 WT and AAMN1 strain for each GLP1-GIP and GLP1-GIP precursor molecule. *p < 0.05, **p < 0.01.

[0045] The figures are schematic and simplified for clarity.

[0046] DESCRIPTION

[0047] In the following detailed description, numerous specific details are set forth to provide a full understanding of the subject technology. It will be apparent to one of ordinary skill in the art that the subject technology may be practised without some of these specific details. Well-known structures and techniques may not be shown in detail so as to not obscure the subject technology.

[0048] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and / or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

[0049] In the following, the elements of the present disclosure will be described. These elements may be listed with specific elements, but it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0050] To facilitate an understanding of the present subject technology, a number of terms and phrases are defined below. Unless defined otherwise herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0051] Definitions

[0052] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4% to 20%" should be interpreted to include not only the explicitly recited values of 4% to 20%, but to also include individual values and subranges within the indicated range Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20% and sub-ranges such as from 4-10 %, 5-15 %, 10-20%, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range, or the characteristics being described.

[0053] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a filter" includes one or more of such different filters and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the present disclosure.

[0054] Typically, a fermentation medium as referred to herein would include two or more of the following media: base medium, fed-batch medium, and continuous feed medium. The base medium comprises 1-10% (w / w) carbon source and 0.1-2% (w / w) nitrogen source. The fed-batch medium or DOS1 comprises 20-50% (w / w) carbon source and 0.1-2% (w / w) nitrogen source. The continuous feed medium or DOS2 comprises 10-40% (w / w) carbon source and 0.1-2% (w / w) nitrogen source. As used herein, “Polypeptide 1” refers to any polypeptide that is an extended semaglutide precursor (extended [Arg34]GLP-1(9-37). Examples of such semaglutide precursors may be found in WO 2015 / 091613. As used herein, “Polypeptide 2” refers to any polypeptide that is an extended or non-extended GLP1-GIP fusion polypeptide precursor. Examples of GLP1-GIP fusion polypeptides may be found in WO 2022 / 018186, such as, e.g. EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS or EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS. Suitable extensions can be found in WO 2015 / 091613. Two examples of an N-terminally extended GLP1-GIP fusion polypeptide precursor is DVRPGHPLEDEGDREGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID No: 6; GLP1-GIP fusion precursor 1 with N-terminal extension 5) and DVRPGQPLHDEGDREGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID No: 7; GLP1-GIP fusion precursor 1 with N-terminal extension 6). GLP1-GIP fusion precursor 1 with N- terminal extension 5 is also referred to as extended_GLP1-GIP_1 in the sequence listing. GLP1- GIP fusion precursor 1 with N-terminal extension 6 is also referred to as extended_GLP1-GIP_2 in the sequence listing

[0055] The term “and / or” wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term.

[0056] The term “aerobic fermentation” is used herein to describe the aerobic metabolic processes of yeast cells, including the citric acid cycle and electron transport chain. This aligns with contemporary usage in the field that extends the traditional definition of fermentation to include aerobic respiration for clarity and consistency. For example, the term is used to describe Saccharomyces cerevisiae aerobic respiration, in line current industry and academic usage.

[0057] It should be understood that the embodiments described herein include "consisting" and / or "consisting essentially of" embodiments. As used herein, except where the context requires otherwise due to express language or necessary implication, the word "comprise", and variations thereof such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or element or step or group of integer or element or step, but not the exclusion of any other integer or element or step or group of integer or element or step. When used herein the term “comprise” can be substituted with the terms “contain” or “include” or sometimes when “have”.

[0058] The term “obtainable” can be used interchangeably with “obtained”.

[0059] The term "about" is used herein to mean approximately, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by 20 percent, up or down (higher or lower), preferably 15 percent, up or down (higher or lower), more preferably within 10 percent, up or down (higher or lower), and most preferably by 5 percent, up or down (higher or lower).

[0060] As used herein, the term “acidophilic strain” relates to strains that produce a target peptide more efficiently at a pH below 5.9. As used herein, the term “neutral strain” relates to strains that produce a target peptide more efficiently at a pH in the range of 6-7.5. As used herein, the term “alkaliphilic strain” is used for strains that produce a target peptide more efficiently a pH above 7.5. For the avoidance of doubt, whether a strain is considered an acidophilic strain or an alkaliphilic strain as defined herein is not based on the strain’s ability to survive or grow in the indicated pH environment but is based on the strain’s efficiency of producing the target peptide.

[0061] As used herein, the term “endogenous” means, with respect to a host cell, that a polypeptide or nucleic acid or gene does naturally occur in the host cell, meaning that a gene which is endogenous to a host cell occurs naturally in said host cell and is native in said host cell.

[0062] The term “fermentation broth” as used herein means the mixture of water, sugars (fermentable carbon sources), dissolved solids (if present), microorganisms producing alcohols, product alcohols and all other constituents of the material in which product alcohol is being made by the reaction of sugars to alcohol, water and carbon dioxide (CO2) by the microorganisms present. From time to time, as used herein the term "fermentation medium" and "fermented mixture" can be used synonymously with "fermentation broth".

[0063] The term “host cell” as used herein means any microbial, fungal, mammalian, or plant cell into which nucleic acid construct or expression vector comprising a target peptide of the present disclosure has been introduced. Preferred host cells are yeast host cells. Methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. In some embodiments, the host cell is an isolated recombinant host ceil that is partially or completely separated from at least one other component with, including but not limited to, proteins, nucleic acids, cells, etc.

[0064] The term "productivity of the strain" is intended to mean its capacity for producing large amounts of a molecule(s) of interest.

[0065] A "fermentation process" describes a sequence of activities comprising the preparation of the fermentation medium and the cultivation of cells in the fermentation medium. "Cultivation of the cells" or "growth of the cells" is not understood to be limited to an exponential growth phase but can also include the physiological state of the cells at the beginning of growth after inoculation and during a stationary phase. The fermentation process can be stopped by appropriate measures that limit or prevent the growth of the cells, for instance but not being limited to reducing the temperature of the fermentation broth.

[0066] The term "complex nutrient source" is used herein for nutrient sources which are composed of chemically undefined compounds, i.e., compounds that are not known by their chemical formula, preferably comprising undefined organic nitrogen- and I or undefined organic carbon containing compounds. In contrast thereto, a "chemically defined nutrient source" (e.g., "chemically defined carbon source" or "chemically defined nitrogen source") is understood to be used for nutrient sources which are composed of chemically defined compounds. A "chemically defined component" is a component which is known by its chemical formula.

[0067] The term "complex nitrogen source" is used herein for a nutrient source that is composed of one or more chemically undefined nitrogen containing compounds, i.e., nitrogen containing compounds that are not known by their chemical formula, preferably comprising organic nitrogen containing compounds, e.g., proteins and / or amino acids with unknown composition. Preferably, the complex nitrogen source comprises one or more chemically undefined nitrogen containing proteins and / or amino acids. Preferably, the complex nitrogen source comprises one or more chemically undefined nitrogen containing proteins. An example of a complex nitrogen source is yeast extract. Yeast extract is a complex mixture of nutrients, such as amino acids, peptides, vitamins, and minerals, that is commonly used as a supplement in the fermentation of Saccharomyces cerevisiae (S. cerevisiae).

[0068] The term "complex carbon source" is used herein for a carbon source that is composed of one or more chemically undefined carbon containing compounds, i.e., carbon containing compounds that are not known by their chemical formula, preferably comprising organic carbon containing compounds, e.g., carbohydrates with unknown composition.

[0069] The term "adjusting the pH of the fermentation medium" can mean adjusting the pH of the fermentation medium either by the addition of an acid and / or a base or can mean adjusting the pH of the fermentation medium by choosing the medium components to yield the desired pH after mixing of all medium components.

[0070] The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide that is operably linked to one or more control sequences that provides for the expression of the polynucleotide.

[0071] The term “gene” as used herein refers to a nucleic acid (polynucleotide) that encodes and directs the expression of a protein or RNA. The term “parental cell” or “parental strain” refers to “unmodified” parental cells. For example, a “parental cell” refers to any cell (or strain) in which the genome of the “parental” cell is modified (e.g. via a genetic modification introduced into the parental cell) to generate a variant (daughter) cell or strain, such that the “parental” and “daughter” cell (or strain) differ.

[0072] The term “variant cell” or “variant strain” refer to the modified cell or strain that is derived (i.e., obtained from or obtainable) from a parental (or reference) strain or cell, wherein the variant cell or strain comprises a genetic modification which is not present in the parental strain, such that, by comparison, phenotype differences between the “parental” and “variant” strains can be attributed to the genetic modification. In other terms, parental and variant stains are otherwise isogenic except for the genetic modification(s) “introduced” into the variant strain.

[0073] Thus, parental and variant “cells” or “strains” can be described has having certain characteristics, such as genetic modifications, expression phenotypes, morphology phenotypes and the like; however, the skilled person will appreciate that it is technically the “cells” of the parental or variant strain that have such characteristics, and the “strains” are referred to for convenience.

[0074] The term “target peptide”, as used herein, includes polypeptides and proteins. Preferred target peptides of the disclosure are “polypeptide 1” and “polypeptide 2” as defined herein. More particular, examples of target peptides of the disclosure are extended [Arg34]GLP- 1(9-37), extended [Arg34]GLP-1(7-37), GLP1-GIP fusion polypeptides, and extended GLP1-GIP fusion polypeptides. By extended semaglutide precursor we mean N-terminally extended [Arg34]GLP-1(9-37). Examples of such extended semaglutide precursors can be found in WO 2015 / 091613. By extended GLP1- GIP fusion polypeptide precursor we mean N-terminally extended GLP1-GIP fusion polypeptide. Examples of such GLP1-GIP fusion polypeptides can be found in WO 2022 / 018186. Specific examples of GLP1-GIP fusion polypeptides are

[0075] EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID No: 4) and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID No: 5). Suitable extensions can be found in WO 2015 / 091613.

[0076] The terms “polypeptide” and “protein” (and / or their respective plural forms), as used herein, are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one-letter or three-letter codes for amino acid residues are used herein. The polymer can be linear or branched. It can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention: for example, disulfide bond formation, glycosylation, lipidation. acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural ammo acids, etc,), as well as other modifications known in the art.

[0077] Herein “Pichia pastoris” is used synonymously for all, Komagataella pastoris, Komagataella phaffii and Komagataella pseudopastoris.

[0078] Viscosity is a measure of the resistance of a fluid to deformation by mechanical stress, such as shear stress or tensile stress. As used herein, the term “viscosity” is used interchangeably with viscous fluid behaviour. As an example “pseudoplastic viscosity” refers to the pseudoplastic behaviour of a viscous fluid.

[0079] The term "viscosity" or "culture broth viscosity" means the dynamic or absolute viscosity of the culture broth being formed by cultivation of the host cells in cultivation medium. The term “viscosity” also covers inter alia “product-related viscosity” and “cell-derived viscosity”. In the present context, viscosity can also refer to the resistance of a cell broth comprising yeast cells to mechanical stress, such as e.g., as provided by a rotor / impeller. Because the viscosity of a cell broth can be difficult to measure directly, indirect measurements of viscosity can also be used, such as the dissolved oxygen content of the culture broth at a pre-selected amount of agitation, the amount of agitation required to maintain a preselected dissolved oxygen content, the amount of power required to agitate a cell broth to maintain a preselected dissolved oxygen content.

[0080] The term “soluble fraction” as used herein refers to the portion of a fermentation broth that remains in solution after removal of any insoluble materials, such as cells or other debris. This fraction contains all the soluble components of the broth, including sugars, amino acids, peptides, proteins, and other small molecules.

[0081] With regard to the term “pH setpoint”, it will be understood that the actual process pH value may vary in the range of +0.05 to -0.15 pH units relative to the indicated pH setpoint value.

[0082] Detailed description

[0083] High viscosity of a fermentation broth during the recombinant production of target peptides can pose significant challenges for the reproducible production of high-quality target-peptides. High fermentation broth viscosity can lead to poor mixing and aeration, resulting decreased mass transfer of nutrients, oxygen, and waste products, which can negatively impact cell growth and product yield. Also, or alternatively, high viscosity can reduce the efficiency of liquid mixing, leading to spatial variations in key fermentation parameters, such as pH, temperature, and substrate concentration. These spatial inhomogeneities can negatively impact fermentation performance. Also, or alternatively, mass transfer rates may be reduced at higher liquid viscosities. In aerobic fermentation, oxygen is typically provided by injecting or sparging air, oxygen, or mixtures thereof into the fermenter. However, high viscosity can hinder oxygen transfer from the gas phase to the liquid phase, which may decrease productivity and yield. Additionally, high viscosity may result in poor carbon dioxide transfer from the broth to the gas phase. High concentrations of carbon dioxide in the fermentation liquid can be toxic to the microorganisms producing the desired polypeptide, negatively affecting productivity and yield.

[0084] Different types of viscosities may be present in a fermentation broth. A proper understanding of the different types of viscosities present in a fermentation broth is crucial for an efficient fermentation process

[0085] Different types of high viscosity fluid behaviour may be present in a fermentation broth. A proper understanding of the nature of the viscosity in a fermentation broth is crucial for an efficient fermentation process. Examples of such different types of high viscosity fluid behaviour are separated into two main categories, i.e. Newtonian and non - Newtonian. In the case of Newtonian, the viscosity remains constant regardless of the applied shear rate. Fluids that show a nonNewtonian behaviour, such as, e.g., pseudoplastic behaviour or dilitant behaviour, are the most challenging ones. A pseudoplastic behaviour can lead to viscosity decreasing with increased shear rate and dilitant or shear thickening behaviour can lead to viscosity increasing with increased shear rate. The former is responsible for significant substrate and oxygen gradients as high shear areas in the bioreactor, e.g. impellers typically exhibit low viscosity, whereas low shear regions far away from the stirrer and buffles tend to have very high viscosities. The opposite would be expected in the case of fermentation broth showing dilitant behaviour. In addition, the high viscosity at the vicinity of the impellers will significantly increase the power consumption. Non-Newtonian fermentation broth of high viscosity in general will result in non-efficient processes, due to their impact on various factors such as gas-liquid mass transfer, heat transfer, the appropriate distribution of feeds, the local concentration of substrate and oxygen and power consumption for agitation, the hindrance of the efficient dissipation of metabolic and mechanically-induced heat and ultimately result in lower product yields and increased cost of operation.

[0086] The inventors surprisingly found that the viscosity showing pseudoplastic and thixotropic characteristics originated from aggregates forming during fermentation which, however, after centrifugation are not part of the pellet but part of the soluble fraction. This type of viscosity is referred to herein as “product-related viscosity” because the aggregates are formed by the target peptide, such as, e.g., a GLP-1 peptide or precursor or fusion thereof. By this unexpected finding the inventors could control the viscosity by adjusting the pH to a range where the target peptide would not form aggregates. Further to the surprising finding that the aggregates are part of the soluble fraction, the inventors found that, even if the fermentation was conducted at a pH where the target peptide forms aggregates due to the use of an acidophilic strain or host cell, the recovery rate could be improved by increasing the pH only after clarification instead of prior to clarification. Increasing the pH after clarification avoids cell lysis during alkalic treatment and thus reduced the release of host cell proteins (HCP), which are an undesirable side-product, because of the difficulty of removing HCP during downstream process steps.

[0087] The present inventors further surprisingly found that the AMN1 gene in the host cell used for the recombinant production of the target peptide is the root cause of the viscosity of dilatant nature, which is referred to herein as “cell-derived viscosity’’. The inventors found that the deletion or knockout of the AMN1 gene led to a significant reduction in viscosity of the fermentation broth. More particularly, the inventors surprisingly found that the deletion of the AMN1D368allele or the modification of the AMN1D368allele to an AMN1D3B8Vallele led to a signification reduction in viscosity of the fermentation broth compared to the viscosity of the fermentation broth when using the parental host cell comprising the AMN1D368allele.

[0088] By identifying the root causes, the present inventors could tackle the viscosity problem and / or increase the recovery rate.

[0089] Fermentation process

[0090] Typically, aggregation is linked to precipitation. Surprisingly, the target peptides described herein stay in the soluble fraction when aggregated leading to an increase in viscosity of the fermentation broth. This unexpected finding resulted in waiting with adjusting the pH of the fermentation broth to re-solubilise and de-aggregate the product until after clarification.

[0091] In a first aspect, the present disclosure is directed to a fermentation process for expressing a target peptide, such as, e.g., an N-terminally extended [Arg34]GLP-1(9-37) or an extended GLP1-GIP fusion polypeptide precursor, comprising the steps of adjusting the pH of an inoculated fermentation medium to a desired pH setpoint, such as a pH setpoint in the range of 3.0-6.0, clarifying the fermentation broth to obtain a soluble fraction and adjusting the pH of the soluble fraction to a pH in the range of 7.5 to 12.0 to recover the target peptide.

[0092] Thus, in one embodiment, the fermentation process for expression of a target peptide comprises the steps of a. preparing a fermentation medium, b. inoculating the fermentation medium obtainable in step a with a yeast cell comprising a DNA sequence encoding said target peptide, c. adjusting the pH of the inoculated fermentation medium obtainable in step b. to a desired pH setpoint, wherein the pH setpoint is in the range of 3.0-6.0, and keeping the pH constant at said pH setpoint, d. cultivating the inoculated fermentation medium obtainable in step c. at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth, e. harvesting the fermentation broth, f. clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation, g. recovering the target peptide by adjusting the pH of the soluble fraction obtainable in step g. to a pH value in the range of 7.5 to 12.0, and optionally h. purifying the target peptide recovered in step g.

[0093] In particular embodiment, the target peptide is an N-terminally extended [Arg34]GLP-1(9- 37), a GLP1-GIP fusion polypeptide or an extended GLP1-GIP fusion polypeptide. Examples of extended GLP1-GIP fusion polypeptide are EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS. Suitable extensions can be found in WO 2015 / 091613.

[0094] In one embodiment, the fermentation medium of the present disclosure comprises medium components required for the growth of a cultivated cell. In one embodiment, the fermentation medium comprises one or more components selected from the group consisting of nitrogen source, phosphor source, sulfur source and salt, and optionally one or more further components selected from the group consisting of micronutrients, like vitamins, amino acids, minerals, and trace elements. In one embodiment, the fermentation medium also comprises a carbon source. Such components are generally known in the art, see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989 Cold Spring Harbor, N. Y.; A.K. Seresht, et al. Biotechnology and Bioengineering 2013, 110(10), 2749-2763.

[0095] In one embodiment, the fermentation broth comprises a complex nutrient source, optionally wherein the complex nutrient source is a complex nitrogen source. Complex sources of nitrogen can include, but are not limited to protein-containing substances, such as an extract from microbial, animal or plant cells. In one embodiment, the fermentation medium comprises a defined nitrogen source. The defined nitrogen source may be an inorganic nitrogen source such as, e.g., ammonia, ammonium, ammonium salts, urea, nitrate, nitrate salts, nitrite, amino acids, and combinations thereof.

[0096] In one embodiment, the fermentation medium comprises a carbon source. Exemplary carbon sources include but are not limited to glucose, glycerol, sucrose, molasses, and mixtures thereof.

[0097] In one embodiment, the fermentation medium comprises a carbon source and / or a nitrogen source.

[0098] Oxygen is usually provided during the cultivation of the cells by aeration of the fermentation medium by stirring and gassing. Hydrogen is usually provided due to the presence of water in the aqueous fermentation medium. However, hydrogen and oxygen are also contained within the carbon and / or nitrogen source and can be provided that way.

[0099] Preferably, prior inoculation the fermentation medium and feed solutions are sterilised in order to prevent or reduce growth of unwanted microorganisms during the fermentation process. Sterilisation can be performed with methods known in the art, for example but not limited to heat sterilisation or sterile filtration. Medium components can be sterilised separately from other medium components to avoid interactions of medium components during sterilization treatment or to avoid decomposition of medium components under sterilisation conditions.

[0100] In some embodiments, the yeast cell is an acidophilic yeast cell. In some embodiments, the yeast cell has a higher expression yield or recovery rate of target peptide under acidic process conditions than under basic process conditions. In some embodiments, the yeast cell produces the target peptide more efficiently at a pH below 5.9 compared to a pH above 5.9. In some embodiments, the yeast cell produces the target peptide more efficiently at a pH in the range of 4.5 to 5.9. The skilled person will understand that, e.g. the cell may still grow at a pH outside the pH range that is optimal for recovery yield, however the yeast cell may produce a target peptide inefficiently or not at all.

[0101] In some embodiments, the yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum. In particular embodiments, the yeast cell is selected from the group consisting of Saccharomyces cerevisae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia. In preferred embodiments, the yeast cell is Saccharomyces cerevisae or Pichia pastoris. In particularly preferred embodiments, the yeast cell is a Saccharomyces cell, preferably a Saccharomyces cerevisiae cell. In some embodiments, the yeast cell is a Komagataella cell, preferably a Komagataella phaffii, Komagataella pastoris or Komagataella ulmi cell.

[0102] In some embodiments, the fermentation process is a submerged fermentation process. In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0103] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0104] Zi is a polypeptide comprising at least 2 amino acid residues;

[0105] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0106] X5 is selected from the genetically encoded amino acids but S and I;

[0107] X6is absent or selected from the genetically encoded amino acids; and

[0108] Z2is an optional polypeptide or amino acid residue.

[0109] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0110] Z2-X6-X5-X4-G-D-R-Z1 (I) SEQ ID NO: 1 wherein

[0111] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0112] X4 is E, Q, L, G, or A;

[0113] X5is selected from the genetically encoded amino acids but S and I;

[0114] Xe is absent or selected from the genetically encoded amino acids; and

[0115] Z2is an optional polypeptide or amino acid residue.

[0116] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0117] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0118] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0119] X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE;

[0120] Xe is absent or selected from the genetically encoded amino acids; and

[0121] Z2is an optional polypeptide or amino acid residue. In some embodiments, Z2is a polypeptide facilitating the expression of said Enterokinase-cleavable fusion polypeptide in a yeast cell. In some embodiments, Z2is a polypeptide having from 2 to 50 amino acid residues. In some embodiments, Z2consist of one amino acid or is absent.

[0122] In some embodiments, Zi comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme. In some particular embodiments, Zi is a GLP-1 peptide or a functional variant thereof, such as K34R-GLP-1(7-37) or K34R-GLP-1(9-37). In some embodiments, Zi is a glucagon peptide or a functional variant thereof. In some embodiments, Zi is a GLP-1 -GIP fusion polypeptide or a functional variant thereof, such as EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS or EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS. In some embodiments, Zi is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof, such as an extended [Glu14, Arg17, Pro37]-pramlintide.

[0123] In some embodiments, the fermentation medium is prepared by mixing in water components supporting growth of the yeast cell. In some embodiments, the fermentation medium comprises a base medium, a fed-batch medium, and / or a continuous feed medium. In some embodiments, the base medium comprises 1-10% (w / w) carbon source and 0.1-2% (w / w) nitrogen source. In some embodiments, the fed-batch medium or DOS1 comprises 20-50% (w / w) carbon source and 0.1-2% (w / w) nitrogen source. In some embodiments, the continuous feed medium or DOS2 comprises 10- 40% (w / w) carbon source and 0.1-2% (w / w) nitrogen source.

[0124] In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.6 -12. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.7 -12. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.8 -12. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.9 -12. In some embodiments, the pH in step h is adjusted to a pH value in the range of 8.0 -12. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.5. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.4. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.3. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.2. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.1. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -11.0. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.9. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.8. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.7. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.6. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.5. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.4. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.3. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.2. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.1. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -10.0. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.9. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.8. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.7. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.6. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.5. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.4. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.3. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.2. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.1. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -9.0. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -8.9. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -8.8. In some embodiments, the PH in step h is adjusted to a PH value in the range of 7.5 -8.7. In some embodiments, the PH in step h is adjusted to a PH value in the range of 7.5 -8.6. In some embodiments, the pH in step h is adjusted to a pH value in the range of 7.5 -8.5.

[0125] In some embodiments, the target peptide is recovered in at least 0.2-10 g / L / day.

[0126] In a second aspect, the present disclosure relates to a method for producing a target peptide. The method for producing a target peptide comprises the steps of transforming a genetically modified yeast cell with a polynucleotide vector encoding a target peptide, wherein the AMN1 gene of the yeast cell has been deleted or modified or disrupted, and inoculating a fermentation medium with the transformed yeast cell. The inoculated fermentation medium can be adjusted to a desired pH setpoint, wherein the pH setpoint is above 5.5. The pH is then typically kept constant at said pH setpoint. The inoculated fermentation medium can be cultivated at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth to then harvest the fermentation broth. Afterwards the fermentation broth can be clarified to obtain a supernatant, optionally by centrifugation, and recover the target peptide. The recovered target peptide may be purified.

[0127] In some embodiments, the method for producing a target peptide comprises the steps of a) transforming a genetically modified yeast host cell with a polynucleotide vector encoding a target peptide or a precursor thereof, wherein the AMN1D368allele of the yeast host cell has been deleted or disrupted or has been modified, b) inoculating a fermentation medium with the transformed yeast cell obtainable in step a, c) adjusting the pH of the inoculated fermentation medium obtainable in step b to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint, d) cultivating the inoculated fermentation medium obtainable in step c. at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth, e) harvesting the fermentation broth, f) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation, g) recovering the target peptide, and optionally h) purifying the target peptide recovered in step g.

[0128] In particular embodiments, the method for producing a target peptide comprises the steps of a) providing a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele; b) deleting the D368 allele or modifying the D368 allele into an D368V allele to obtain a variant Saccharomyces cerevisiae cell; c) transforming the variant Saccharomyces cerevisiae cell with a polynucleotide vector encoding a target peptide or a precursor thereof; d) inoculating a fermentation medium with the transformed variant Saccharomyces cerevisiae cell obtainable in step c; e) adjusting the pH of the inoculated fermentation medium obtainable in step d) to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint; f) cultivating the inoculated fermentation medium obtainable in step e) at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth; g) harvesting the fermentation broth; h) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation; i) recovering the target peptide; and optionally h) purifying the target peptide recovered in step g.

[0129] In some embodiments, the pH setpoint is in the range of 5.5-9.4. In some embodiments, the pH setpoint is in the range of 5.5-9.3. In some embodiments, the pH setpoint is in the range of 5.5-9.2. In some embodiments, the pH setpoint is in the range of 5.5-9.1. In some embodiments, the pH setpoint is in the range of 5.5-9.0. In some embodiments, the pH setpoint is in the range of 5.5-8.9. In some embodiments, the pH setpoint is in the range of 5.5-8.8. In some embodiments, the pH setpoint is in the range of 5.5-8.9. In some embodiments, the pH setpoint is in the range of 5.5-8.7. In some embodiments, the pH setpoint is in the range of 5.5-8.6. In some embodiments, the pH setpoint is in the range of 5.5-8.5. In some embodiments, the pH setpoint is in the range of 5.5-8.4. In some embodiments, the pH setpoint is in the range of 5.5-8.3. In some embodiments, the pH setpoint is in the range of 5.5-8.2. In some embodiments, the pH setpoint is in the range of 5.5-8.1. In some embodiments, the pH setpoint is in the range of 5.5-8.0. In some embodiments, the pH setpoint is in the range of 5.5-7.9. In some embodiments, the pH setpoint is in the range of 5.5-7.8. In some embodiments, the pH setpoint is in the range of 5.5-7.7. In some embodiments, the pH setpoint is in the range of 5.5-7.6. In some embodiments, the pH setpoint is in the range of 5.5-7.5. In some embodiments, the pH setpoint is in the range of 5.5-7.4. In some embodiments, the pH setpoint is in the range of 5.5-7.3. In some embodiments, the pH setpoint is in the range of 5.5-7.2. In some embodiments, the pH setpoint is in the range of 5.5-7.1. In some embodiments, the pH setpoint is in the range of 5.5-7.0. In some embodiments, the pH setpoint is in the range of 5.5-6.9. In some embodiments, the pH setpoint is in the range of 5.6-9.4. In some embodiments, the pH setpoint is in the range of 5.7-9.4. In some embodiments, the pH setpoint is in the range of 5.8-9.4. In some embodiments, the pH setpoint is in the range of 5.9-9.4. In some embodiments, the pH setpoint is in the range of 6.0-9.4. In some embodiments, the pH setpoint is in the range of 6.1-9.4. In some embodiments, the pH setpoint is in the range of 6.2-9.4.

[0130] In some embodiments, the yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum. In particular embodiments, the yeast cell is selected from the group consisting of Saccharomyces cerevisae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia. In preferred embodiments, the yeast cell is Saccharomyces cerevisae or Pichia pastoris. In particularly preferred embodiments, the yeast cell is a Saccharomyces cell, preferably a Saccharomyces cerevisiae cell. In some embodiments, the yeast cell is a Komagataella cell, preferably a Komagataella phaffii, Komagataella pastoris or Komagataella ulmi cell.

[0131] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0132] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0133] Zi is a polypeptide comprising at least 2 amino acid residues;

[0134] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0135] X5is selected from the genetically encoded amino acids but S and I;

[0136] X6is absent or selected from the genetically encoded amino acids; and

[0137] Z2is an optional polypeptide or amino acid residue.

[0138] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0139] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0140] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0141] X4 is E, Q, L, G, or A;

[0142] X5 is selected from the genetically encoded amino acids but S and I;

[0143] X6is absent or selected from the genetically encoded amino acids; and

[0144] Z2is an optional polypeptide or amino acid residue.

[0145] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0146] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0147] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0148] X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE;

[0149] X6is absent or selected from the genetically encoded amino acids; and Z2is an optional polypeptide or amino acid residue.

[0150] In some embodiments, Z2is a polypeptide facilitating the expression of said Enterokinase-cleavable fusion polypeptide in a yeast cell. In some embodiments, Z2is a polypeptide having from 2 to 50 amino acid residues. In some embodiments, Z2consists of one amino acid or is absent.

[0151] In some embodiments, Zi comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme. In some particular embodiments, Zi is a GLP-1 peptide or a functional variant thereof, such as K34R-GLP-1(7-37) or K34R-GLP-1(9-37). In some embodiments, Zi is a glucagon peptide or a functional variant thereof. In some embodiments, Zi is a GLP-1 -GIP fusion polypeptide or a functional variant thereof, such as, e.g.

[0152] EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS or

[0153] EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS. In some embodiments, Zi is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof, such as extended [Glu14, Arg17, Pro37]-pramlintide.

[0154] Variant yeast cell

[0155] In a third aspect, the present disclosure relates to a variant yeast cell derived from a parental cell comprising an endogenous gene encoding Amn1 protein, wherein the variant yeast cell comprises a deletion or disruption or modification in an endogenous gene encoding Amn1 protein, and wherein the variant yeast cell further comprises a polynucleotide vector encoding a target peptide. The variant yeast cell may be capable of expressing the target peptide in higher yields at a pH above 5.5, such as at a pH in a range of about 5.5-12, than at a pH below 5.5, such as at a pH in range of 5.4-1 .

[0156] In some embodiments, the variant yeast cell is derived from a parental cell comprising an endogenous gene encoding Amn1 protein, wherein the variant yeast cell comprises (a) a deletion or disruption or modification in an endogenous gene encoding Amn1 protein, and wherein (b) the variant yeast cell further comprises a polynucleotide vector encoding a target peptide, and wherein (c) the variant yeast cell is capable of expressing the target peptide in higher yields at a pH above 5.5 than below a pH of 5.5.

[0157] In preferred embodiments, the variant yeast cell is a Saccharomyces cerevisiae yeast cell, wherein the Saccharomyces cerevisiae yeast cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant yeast cell has been modified such that the D368 allele has been knocked out or has been modified to an D368V allele; and wherein the variant yeast cell further comprises a polynucleotide vector encoding a target peptide. In further preferred embodiments, the variant yeast cell is a variant Saccharomyces cerevisiae cell, wherein the variant cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant yeast cell has been modified such that the D368 allele has been knocked out or has been modified to an D368V allele; and wherein the variant cell further comprises a polynucleotide vector encoding a target peptide, wherein the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0158] Z2-X6-X5-X4-G-D-R-Z1 (I) SEQ ID NO: 1 wherein

[0159] Z2is an optional polypeptide or amino acid residue;

[0160] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0161] X5 is selected from the genetically encoded amino acids but S and I;

[0162] Xe is absent or selected from the genetically encoded amino acids; and

[0163] Z1 is selected from the group consisting of K34R-GLP-1(7-37), K34R-GLP-1(9- 37), EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS, and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS.

[0164] In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-12 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-11 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-10 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-9.5 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-9.0 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.9 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.8 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.7 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.6 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.5 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.4 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.3 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.2 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.1 than at a pH in the range of 5.4-1.0. In some embodiments, the variant yeast cell is capable of expressing the target peptide in higher yields at pH in the range of 5.5-8.0 than at a pH in the range of 5.4-1.0.

[0165] In some embodiments, the variant yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum. In particular embodiments, the variant yeast cell is selected from the group consisting of Saccharomyces cerevisae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia. In some embodiments, the yeast cell is a Komagataella cell, preferably a Komagataella phaffii, Komagataella pastoris or Komagataella ulmi cell. In preferred embodiments, the variant yeast cell is Saccharomyces cerevisiae or Pichia pastoris. In particularly preferred embodiments, the variant yeast cell is a Saccharomyces cell, preferably a Saccharomyces cerevisiae cell.

[0166] In further preferred embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0167] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0168] Zi is a polypeptide comprising at least 2 amino acid residues;

[0169] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0170] X5is selected from the genetically encoded amino acids but S and I;

[0171] X6is absent or selected from the genetically encoded amino acids; and

[0172] Z2is an optional polypeptide or amino acid residue.

[0173] In some embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0174] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0175] Zi is a polypeptide comprising at least 2 amino acid residues;

[0176] X4 is E, Q, L, G, or A;

[0177] X5is selected from the genetically encoded amino acids but S and I;

[0178] X6is absent or selected from the genetically encoded amino acids; and

[0179] Z2 is an optional polypeptide or amino acid residue.

[0180] In particularly preferred embodiments, the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0181] Z2-X6-X5-X4-G-D-R-Z1 (I) SEQ ID NO: 1 wherein

[0182] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0183] X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE;

[0184] Xe is absent or selected from the genetically encoded amino acids; and

[0185] Z2is an optional polypeptide or amino acid residue.

[0186] In some embodiments, Z2is a polypeptide facilitating the expression of said Enterokinase-cleavable fusion polypeptide in a yeast cell. In some embodiments, Z2is a polypeptide having from 2 to 50 amino acid residues. In some embodiments, Z2consist of one amino acid or is absent.

[0187] In some embodiments, Z1 comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme. In some particular embodiments, Z1 is a GLP-1 peptide or a functional variant thereof, such as K34R-GLP-1(7-37) or K34R-GLP-1(9-37). In some embodiments, Z1 is a glucagon peptide or a functional variant thereof. In some embodiments, Z1 is a GLP-1 -GIP fusion polypeptide or a functional variant thereof, such as, e.g. EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS or EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS. In some embodiments, Z1 is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof, such as extended [Glu14, Arg17, Pro37]-pramlintide.

[0188] In some embodiments, the optimal pH for expressing the target peptide is at a pH range of 6.0-8.5. Uses of a yeast cell comprising a deletion or modification

[0189] In a fourth aspect, the present disclosure relates to the use of a yeast cell, such as a recombinant yeast cell, wherein the yeast cell comprises a deletion or disruption or modification in a gene encoding Amn1 protein for the recombinant production of a target peptide.

[0190] Particular embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been deleted. Further or alternative embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1-GIP peptide or a functional variant thereof, wherein the AMN1D3S8allele has been modified such that the AMN1D368allele has been deleted.

[0191] Further particular embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele. Further alternative or particular embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1-GIP peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele.

[0192] In some embodiments, the yeast cell is a variant yeast cell according to the third aspect of the disclosure. Particularly preferred embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell according to the third aspect of the disclosure, for the recombinant production of a GLP-1 peptide or a functional variant thereof. Further particularly preferred embodiments relate to the use of a variant Saccharomyces cerevisiae yeast cell according to the third aspect of the disclosure, for the recombinant production of a GLP-1 peptide or a functional variant thereof.

[0193] In some embodiments, the use of the yeast cell is for reducing or lowering viscosity during fermentation.

[0194] List of embodiments

[0195] Embodiment 1. A fermentation process for expressing a target peptide comprising the steps of a. preparing a fermentation medium, b. inoculating the fermentation medium of step a with a yeast cell comprising a DNA sequence encoding said target peptide, c. adjusting the pH of the inoculated fermentation medium obtainable in step b. to a desired pH setpoint, wherein the pH setpoint is in the range of 3.0-6.0, and keeping the pH constant at said pH setpoint, d. cultivating the inoculated fermentation medium obtainable in step c. at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth, e. harvesting the fermentation broth, f. clarifying the fermentation broth to obtain a supernatant optionally by centrifugation, g. recovering the target peptide by adjusting the pH of the supernatant obtainable in step g to a pH value in the range of 7.5 to 12.0, and optionally h. purifying the target peptide recovered in step g.

[0196] Embodiment 2. The fermentation process according to embodiment 1 , wherein the yeast cell has a higher expression yield at a pH below 5.9 compared to a pH above 5.9.

[0197] Embodiment 3. The fermentation process according to embodiment 1 or embodiment 2, wherein the yeast cell is an acidophilic yeast cell.

[0198] Embodiment 4. The fermentation process according to any one of embodiments 1 to 3, wherein the fermentation process is a submerged fermentation process.

[0199] Embodiment 5. The fermentation process according to any one of embodiments 1 to 4, wherein the yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum.

[0200] Embodiment 6. The fermentation process according to any one of embodiments 1 to 5, wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia

[0201] Embodiment 7. The fermentation process according to any one of embodiments 1 to 6, wherein the yeast cell is a Saccharomyces cell or a Pichia pastoris, preferably a Saccharomyces cerevisiae cell.

[0202] Embodiment 8. The fermentation process according to any one of the embodiments 1 to 6, wherein the yeast cell is a Komagataella cell, preferably a Komagataella phaffii, Komagataella pastoris or Komagataella ulmi cell.

[0203] Embodiment 9. The fermentation process according to any one of embodiments 1 to 8, wherein the target peptide is a GLP1-GIP polypeptide, such as EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or

[0204] EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

[0205] Embodiment 10. The fermentation process according to any one of embodiments 1 to 9, wherein the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0206] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0207] Zi is a polypeptide comprising at least 2 amino acid residues;

[0208] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0209] X5 is selected from the genetically encoded amino acids but S and I;

[0210] X6is absent or selected from the genetically encoded amino acids; and

[0211] Z2is an optional polypeptide or amino acid residue.

[0212] Embodiment 11. The fermentation process according to any one of embodiments 1 to 10, wherein the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0213] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0214] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0215] X4 is E, Q, L, G, or A;

[0216] X5is selected from the genetically encoded amino acids but S and I;

[0217] X6is absent or selected from the genetically encoded amino acids; and

[0218] Z2is an optional polypeptide or amino acid residue.

[0219] Embodiment 12. The fermentation process according to embodiment 10 or embodiment 11 , wherein X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE.

[0220] Embodiment 13. The fermentation process according to any one of embodiments 10 to 12, wherein Z2is a polypeptide facilitating the expression of said Enterokinase-cleavable fusion polypeptide in a yeast cell.

[0221] Embodiment 14. The fermentation process according to any one of embodiments 10 to 13, wherein Z2is a polypeptide having from 2 to 50 amino acid residues. Embodiment 15. The fermentation process according to any one of embodiments 10 to 14, wherein Z2consist of one amino acid or is absent.

[0222] Embodiment 16. The fermentation process according to any one of embodiments 10 to 15, wherein Zi comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme.

[0223] Embodiment 17. The fermentation process according to any one of embodiments 10 to 16, wherein Zi is a GLP-1 peptide or a functional variant thereof, such as K34R-GLP-1 (7-37) or K34R-GLP-1(9- 37).

[0224] Embodiment 18. The fermentation process according to any one of embodiments 10 to 17, wherein Zi is a glucagon peptide or a functional variant thereof.

[0225] Embodiment 19. The fermentation process according to any one of embodiments 10 to 18, wherein Zi is a GLP-1-GIP fusion polypeptide or a functional variant thereof, optionally wherein Zi is EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS or

[0226] EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS.

[0227] Embodiment 20. The fermentation process according to any one of embodiments 10 to 18, wherein Zi comprises or consists of EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 2).

[0228] Embodiment 21. The fermentation process according to any one of embodiments 10 to 20, wherein Zi is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof, such as extended [Glu14, Arg17, Pro37]-pramlintide.

[0229] Embodiment 22. The fermentation process according to any one of embodiments 1-16, wherein the target peptide or peptide precursor is selected from the group consisting of GLP-1, amylin, insulin peptides or fusions thereof.

[0230] Embodiment 23. The fermentation process according to any one of embodiments 1 to 22, wherein the fermentation medium is prepared by mixing in water components supporting growth of the yeast cell.

[0231] Embodiment 24. The fermentation process according to any one of embodiments 1 to 23, wherein the fermentation medium is prepared by mixing in water components supporting growth of the yeast cell, and optionally wherein the fermentation medium comprises a based medium, a fed-batch medium, and / or a continuous feed medium.

[0232] Embodiment 25. The fermentation process according to any one of embodiments 1 to 24, wherein the pH in step h is adjusted to a pH value in the range of 7.6 -12, such as in the range of 7.7-12, such as in the range of 7.8-12, such as in the range of 7.9-12, such as in the range of 8.0-12. Embodiment 26. The fermentation process according to any one of embodiments 1 to 24, wherein the pH in step h is adjusted to a pH value in the range of 7.5-11.5, such as in the range of 7.5-11 , such as in the range of 7.5-10.5, such as in the range of 7.5-10.0.

[0233] Embodiment 27. The fermentation process according to any one of embodiments 1 to 24, wherein the pH in step h is adjusted to a pH value in the range of 7.5-9.5, such as in the range of 7.5-9.0, such as in the range of 7.5 to 8.5.

[0234] Embodiment 28. The fermentation process according to any one of embodiments 1 to 27, wherein the target peptide is recovered in at least 0.2-10 g / L / day.

[0235] Embodiment 29. The fermentation process according to embodiment 27 or embodiment 28, wherein the transformed cells grow or are grown under conditions that allow for expression of the target peptide.

[0236] Embodiment 30. A method for producing a target peptide comprising the steps of a) transforming a genetically modified yeast host cell having a AMN1 gene with a polynucleotide vector encoding a target peptide or a precursor thereof, wherein the D368 allele of the AMN1 gene has been modified such that the D368 allele has been modified to a D368V allele of the yeast host cell has been deleted or has been modified or has been disrupted, b) inoculating a fermentation medium with the transformed yeast cell obtainable in step a, c) adjusting the pH of the inoculated fermentation medium obtainable in step b to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint, d) cultivating the inoculated fermentation medium obtainable in step c. at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth, e) harvesting the fermentation broth, f) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation, g) recovering the target peptide, and optionally h) purifying the target peptide recovered in step g.

[0237] Embodiment 31. A method for producing a target peptide comprising the steps of a) providing a variant yeast cell, wherein the yeast cell is derived from a parental cell comprising an endogenous gene encoding Amn1 protein, wherein the variant yeast cell comprises a deletion or modification in the endogenous gene encoding Amn1 protein, and wherein the variant yeast cell further comprises a polynucleotide vector encoding a target peptide, b) inoculating a fermentation medium with the variant yeast cell obtainable in step a, c) adjusting the pH of the inoculated fermentation medium obtainable in step b to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint, d) cultivating the inoculated fermentation medium obtainable in step c. at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth, e) harvesting the fermentation broth, f) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation, g) recovering the target peptide, and optionally h) purifying the target peptide recovered in step g.

[0238] Embodiment 32. A method for producing a target peptide comprises the steps of a) providing a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele; b) deleting the D368 allele or modifying the D368 allele to a D368V allele to obtain a variant Saccharomyces cerevisiae cell; c) transforming the variant Saccharomyces cerevisiae cell obtained in step b with a polynucleotide vector encoding a target peptide or a precursor thereof; d) inoculating a fermentation medium with the transformed variant Saccharomyces cerevisiae cell obtainable in step c; e) adjusting the pH of the inoculated fermentation medium obtainable in step d) to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint; f) cultivating the inoculated fermentation medium obtainable in step e) at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth; g) harvesting the fermentation broth; h) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation; i) recovering the target peptide; and optionally h) purifying the target peptide recovered in step g.

[0239] Embodiment 33. The method of according to any one of embodiments 30-32, wherein the target peptide is a GLP1-GIP polypeptide or an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0240] Z2-X6-X5-X4-G-D-R-Z1 (I) SEQ ID NO: 1 wherein

[0241] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0242] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0243] X5is selected from the genetically encoded amino acids but S and I;

[0244] X6is absent or selected from the genetically encoded amino acids; and

[0245] Z2 is an optional polypeptide or amino acid residue.

[0246] Embodiment 34. The method of according to any one of embodiments 30-32, wherein the target peptide is a GLP1-GIP polypeptide or an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0247] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0248] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0249] X4 is E, Q, L, G, or A;

[0250] X5is selected from the genetically encoded amino acids but S and I;

[0251] X6is absent or selected from the genetically encoded amino acids; and

[0252] Z2is an optional polypeptide or amino acid residue.

[0253] Embodiment 35. The method of according to embodiment 33 or embodiment 34, wherein X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE.

[0254] Embodiment 36. The method of according to any one of embodiments 33-35, wherein Z2is a polypeptide facilitating the expression of said Enterokinase-cleavable fusion polypeptide in a yeast cell, and optionally wherein the Z2 polypeptide comprises or consists of 2 to 50 amino acid residues.

[0255] Embodiment 37. The method of according to any one of embodiments 30-32, wherein the target peptide is a GLP1-GIP polypeptide, such as EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

[0256] Embodiment 38. The method of according to any one of embodiments 33-37, wherein Z2consist of one amino acid or is absent.

[0257] Embodiment 39. The method of according to any one of embodiments 33-38, wherein Zi comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme.

[0258] Embodiment 40. The method of according to any one of embodiments 33-39, wherein Zi is a GLP- 1 peptide or a functional variant thereof, such as K34R-GLP-1(7-37) or K34R-GLP-1(9-37).

[0259] Embodiment 41 . The method of according to any one of embodiments 33-39, wherein Zi is a glucagon peptide or a functional variant thereof.

[0260] Embodiment 42. The method of according to any one of embodiments 33-39, wherein Zi is a GLP- 1-GIP fusion polypeptide or a functional variant thereof.

[0261] Embodiment 43. The method of according to any one of embodiments 33-39, wherein Zi comprises or consists of EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 2).

[0262] Embodiment 44. The method of according to any one of embodiments 33-39, wherein Zi is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof, such as extended [Glu14, Arg17, Pro37]-pramlintide.

[0263] Embodiment 45. The method of according to any one of embodiments 33-39, wherein Zi is EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS or EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS.

[0264] Embodiment 46. The method of according to any one of embodiments 30-45, wherein the pH setpoint is in the range of 5.5-9.5.

[0265] Embodiment 47. The method of any one of embodiments 30 to 46, wherein the pH setpoint is in the range of 5.6-9.0, such as in the range of 5.7-9.0

[0266] Embodiment 48. The method of any one of embodiments 30 to 47, wherein the pH setpoint is in the range of 5.8-9.0, such as in the range of 5.9-9.0.

[0267] Embodiment 49. The method of any one of embodiments 30 to 48, wherein the pH setpoint is in the range of 6.0-9.0.

[0268] Embodiment 50. The method of any one of embodiments 30 to 49, wherein the pH setpoint is in the range of 6.0-8.5, such as in the range of 6.1-8.5.

[0269] Embodiment 51. The method of any one of embodiments 30 to 50, wherein the pH setpoint is in the range of 6.1-8.0, such as 6.1-7.5. Embodiment 52. The method of any one of embodiments 30 to 51 , wherein the pH setpoint is in the range of 6.0-8.0, such as in the range of 6.0-7.5.

[0270] Embodiment 53. The method of any one of embodiments 30 to 52, wherein the pH setpoint is in the range of 6.0-7.0, such as in the range of 6.2-6.8.

[0271] Embodiment 54. The method of any one of embodiments 30 to 53, wherein the yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum.

[0272] Embodiment 55. The method of any one of embodiments 30 to 54, wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia.

[0273] Embodiment 56. The method of any one of embodiments 30 to 55, wherein the yeast cell is Saccharomyces cerevisiae or Pichia pastoris.

[0274] Embodiment 57. The method of any one of embodiments 30 to 56, wherein the target peptide is recovered in at least 0.2-10 g / L / day.

[0275] Embodiment 58. A recombinant yeast cell comprising (a) a deletion or disruption in an endogenous gene encoding Amn1 protein, and wherein said recombinant yeast cell further comprises a polynucleotide vector encoding the GLP-1 peptide or a precursor thereof, and wherein said yeast cell is capable of expressing said GLP-1 peptide or precursor thereof in higher yields at a pH above 5.5 than below a pH 5.5.

[0276] Embodiment 59. The recombinant yeast cell according to embodiment 58, wherein the GLP-1 peptide or precursor thereof is semaglutide or a semaglutide precursor.

[0277] Embodiment 60. The recombinant yeast cell according to embodiment 58 or embodiment 59, wherein the optimal pH for expressing said GLP-1 peptide or precursor is at range of 6.0-8.5.

[0278] Embodiment 61. A variant yeast cell derived from a parental cell comprising an endogenous gene encoding Amn1 protein, wherein the variant yeast cell comprises a deletion or disruption or modification in an endogenous gene encoding Amn1 protein, and wherein the variant yeast cell further comprises a polynucleotide vector encoding a target peptide, and optionally wherein the target peptide is a GLP1-GIP polypeptide, such as EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or

[0279] EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

[0280] Embodiment 62. A variant Saccharomyces cerevisiae cell, wherein the variant cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant cell has been modified such that the D368 allele has been knocked out or has been modified to a D368V allele; and wherein the variant cell further comprises a polynucleotide vector encoding a target peptide, wherein the target peptide is a GLP1-GIP polypeptide or an enterokinase- cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0281] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0282] Z2is an optional polypeptide or amino acid residue;

[0283] X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0284] X5is selected from the genetically encoded amino acids but S and I;

[0285] Xe is absent or selected from the genetically encoded amino acids; and

[0286] Z1 is selected from the group consisting of K34R-GLP-1 (7-37), K34R-GLP-1(9-37), EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS.

[0287] Embodiment 63. The variant yeast cell according to embodiment 61 or embodiment 62, wherein the variant yeast cell is capable of expressing a target peptide in higher yields at a pH above 5.5, such as at a pH in a range of about 5.5-12, than at a pH below 5.5, such as at a pH in a range of 5.4-1.

[0288] Embodiment 64. The variant yeast cell according to embodiment 61 or embodiment 63, wherein the variant yeast cell is selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogateae, Yarrowia, and Geotrichum.

[0289] Embodiment 65. The variant yeast cell according to any one of embodiment 61 or embodiment 63, wherein the variant yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica and Hansenula polymorphia.

[0290] Embodiment 66. The variant yeast cell according to any one of embodiment 61 or embodiment 63, wherein the variant yeast cell is Saccharomyces cerevisiae or Pichia pastoris.

[0291] Embodiment 67. Use of a recombinant yeast cell according to any one of embodiments 58 to 60 or a variant yeast cell according to any one of embodiments 61 to 66 for the recombinant production of a target peptide.

[0292] Embodiment 68. Use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been deleted. Embodiment 69. Use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1-GIP peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been deleted.

[0293] Embodiment 70. Use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or a functional variant thereof, wherein the AMN1D368allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele.

[0294] Embodiment 71. Use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP1-GIP peptide or a functional variant thereof, wherein the AMN1D3S8allele has been modified such that the AMN1D368allele has been modified to an AMN1D368Vallele.

[0295] Embodiment 72. The use according to any one of embodiments 67-71, wherein the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0296] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0297] Zi is a polypeptide comprising at least 2 amino acid residues; 4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;

[0298] X5is selected from the genetically encoded amino acids but S and I;

[0299] X6is absent or selected from the genetically encoded amino acids; and

[0300] Z2 is an optional polypeptide or amino acid residue.

[0301] Embodiment 73. The use according to any one of embodiments 67 to 72, wherein the target peptide is an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):

[0302] Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 wherein

[0303] Z1 is a polypeptide comprising at least 2 amino acid residues;

[0304] X4 is E, Q, L, G, or A;

[0305] X5is selected from the genetically encoded amino acids but S and I;

[0306] X6is absent or selected from the genetically encoded amino acids; and

[0307] Z2is an optional polypeptide or amino acid residue.

[0308] Embodiment 74. The use according to embodiment 72 or embodiment 73, wherein X4-X5 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ, such as DD or DE. Embodiment 75. The use according to any one of embodiments 72 to 74, wherein Z2is a polypeptide facilitating the expression of said enterokinase-cleavable fusion polypeptide in a yeast cell.

[0309] Embodiment 76. The use according to any one of embodiments 72 to 75, wherein Z2is a polypeptide having from 2 to 50 amino acid residues.

[0310] Embodiment 77. The use according to any one of embodiments 72 to 76, wherein Z2consist of one amino acid or is absent.

[0311] Embodiment 78. The use according to any one of embodiments 72 to 77, wherein Zi comprises a functional polypeptide, such as a pharmaceutically active polypeptide or an enzyme.

[0312] Embodiment 79. The use according to any one of embodiments 72 to 78, wherein Zi is a GLP-1 peptide or a functional variant thereof, such as K34R-GLP- 1(7-37) or K34R-GLP-1 (9-37).

[0313] Embodiment 80. The use according to any one of embodiments 72 to 79, wherein Zi is a glucagon peptide or a functional variant thereof.

[0314] Embodiment 81. The use according to any one of embodiments 72 to 80, wherein Zi is a GLP-1-GIP fusion polypeptide or a functional variant thereof.

[0315] Embodiment 82. The use according to any one of embodiments 72 to 81 , wherein Zi comprises or consists of EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 2).

[0316] Embodiment 83. The use according to any one of embodiments 72 to 78, wherein Zi is an amylin peptide or a functional variant thereof, such as cagrilintide or a functional variant thereof.

[0317] Embodiment 84. The use according to any one of embodiments 72 to 78, wherein Zi is EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS or

[0318] EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS.

[0319] Embodiment 85. The use according to embodiment 71 , wherein the target peptide is a GLP1-GIP polypeptide.

[0320] Embodiment 86. The use according to embodiment 69 or 85, wherein the GLP1-GIP polypeptide is EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or

[0321] EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

[0322] Embodiment 87. The use according to any one of embodiments 72 to 86, wherein the use of the cell is for reducing or lowering viscosity during fermentation.

[0323] Embodiment 88. The use according to any one of the embodiments 69-87, wherein the GLP1-GIP polypeptide is EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5). Embodiment 89. The variant cell according to any one of the embodiments 61-67, wherein the GLP1- GIP polypeptide is EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

[0324] Examples

[0325] List of Abbreviations

[0326] PMT Protein mannosyl transferase

[0327] PVDF Polyvinylidene fluoride

[0328] TOF Time-of-flight

[0329] UV Ultra violet

[0330] WT Wild type

[0331] General Materials

[0332] Saccharomyces cerevisiae (S. cerevisiae) strains

[0333] Deletion of FL08 (Liu H, Styles C.A, Fink G.R, Genetics 1996), FLO11 gene (Lo W.S, Dranginis A.M, J Bacterio!, 1996) and AMN~\ gene (Rose M, et al., Yeast. 1995 Jul;11(9):865-71. doi: 10.1002 / yea.320110908. PMID: 7483850) and genomic replacement of the wildtype AMN gene with the A / W / V1D368Vallele (aspartic acid to valine substitution at position 368 of AMN1p sequence) in yeast strains described in the Table above was achieved by homologous recombination using conventional molecular biology and yeast genetic methods (see e.g., Yeast Genetics: Methods and Protocols, Smith J. S, Burke, D.J, editors, 2014). The genetic modifications in the strains were verified by genomic DNA Polymerase Chain Reaction (PCR) and DNA sequencing.

[0334] For simplicity, viscosity is reported in centipoise (cP) herein. It should be noted that 1 cP is equivalent to 1 mPa*s (SI unit). Target peptides:

[0335] Polypeptide 1 is an extended [Arg34]GLP-1(9-37) peptide. Examples of N-terminal extension suitable for [Arg34]GLP-1 (9-37) can be found in WO 2015 / 091613.

[0336] Polypeptide 2 is an extended GLP1-GIP fusion polypeptide. Examples of GLP1-GIP fusion polypeptides can be found in WO 2022 / 018186, e.g.

[0337] EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID No: 4) and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID No: 5). Suitable extensions can be found in WO 2015 / 091613.

[0338] General Methods of Preparation

[0339] Cultivation conditions

[0340] All fermentations were run in continuous mode i.e. the cells are initially cultivated in batch mode, followed by a fed-batch phase triggered by the decrease of the carbon dioxide signal in the off-gas measurement when glucose was depleted during the batch phase. During the fed-batch phase, glucose (50% w / v), (DOS1) is fed into the bioreactor using an exponential feed profile (0.08h’1).

[0341] When the desired biomass is reached, the fed-batch phase is followed by a chemostat or "continuous phase", in which “full medium” (i.e., carbon source, salts, vitamins, and trace metals) (DOS2) is fed into the bioreactor. The dilution rate of the chemostat phase is set at 0.08h’1and the culture volume is controlled via a gravimetric feed. Moreover, base (NH4OH, 10%, ~2.9 M) is added into the bioreactor as needed to maintain a constant pH but also to provide a non-limiting nitrogen source throughout the process.

[0342] The pH setpoint was maintained to 5.3 for the yNN2071 strain and, unless otherwise stated, from 6.2 to 6.5 for the strains of the new host cell lineage.

[0343] General Methods of Detection and Characterisation

[0344] Rheological characterisation

[0345] Rheological characterisation was performed on broth samples withdrawn from the bioreactor. The samples were left at room temperature for approximately 20 minutes and then stirred gently by hand to ensure homogeneity. The viscosity of the samples was measured using a rheometer (micro Vise™, Rheosense). The microVisc measures the pressure drop along an array of sensors when a liquid passes through the cell. The slope (and corresponding R2of the fit) is calculated from the pressure drop as a function of distance (essentially shear stress versus shear rate) and is used to calculate the viscosity. The measurements were performed at a setpoint temperature of 22 °C and at different shear rates (4000, 2000, 1000, 500, 250, 150, 80 s-1where applicable) equipped with a 50pm path microfluidic chip.

[0346] For the rheological analysis of the supernatant and the resuspended pellet, the broth sample was centrifuged at a relative centrifugal force of 2250 x g for 10 minutes to reach pellet-liquid-separation. The supernatant was recovered and the viscosity of the supernatant at different shear rates was measured as described above. The remaining pellet was washed twice with water and brought to the initial weight. Then the viscosity of the so-obtained re-suspended pellet in water was measured at different shear rates as described above.

[0347] The power law model is used to calculate the flow consistency index (K) and flow behaviour index (n) of a fluid. The average flow behaviour index values were determined by integrating the flow behaviour index over the respective time intervals using the composite trapezoidal rule (trapz function from NumPy v1.26) and then dividing it by the duration of those intervals. To implement the power of law model, the shear stress (T) versus shear rate (y) data is plotted on a log-log scale, and the slope and intercept of the linear portion of the resulting curve are used to determine n and K, respectively (described in Stanbhury et al., 2017, chapter 9).

[0348] Off-gas

[0349] The off-gas from all cultures was on-line analysed for oxygen consumption, carbon dioxide and ethanol emission with a Thermo Scientific™ Prima BT Mass Spectrometer.

[0350] Target peptide extraction from cultivation broth

[0351] Dilution method'. 0.1 ml_ of culture are added in 2.7 ml_ de-ionized water, mixed well and centrifuged. 1 ml_ of the supernatant was filtered via a 0.2 pm PVDF filter to a HPLC vial. The dilution method measures the non-aggregated target peptide concentration.

[0352] Alkalic extraction’. 1 mL of culture are added in 4 mL 0.075M phosphate buffer pH 12, mixed well and centrifuged. Supernatant is weighed and diluted 1 :1 with 4N acetic acid and sterile filtered via a 0.2 pm PVDF filter into a HPLC vial. The alkalic extraction measures the total target peptide concentration (both monomeric and aggregated).

[0353] UPLC-MS analysis of the target peptide

[0354] The target peptide was separated and analysed using a reverse phase UPLC-MS (Agilent Technologies 6200 (ESI-TOF LC-MS) on a Poroshell 120 SB-C8 2.1x50 mm column (Agilent), operating at 40 °C and at a flow rate of 0.5 mL / min. Buffer A was composed of 8.8 mM Ammonium Formate + 0.1 % Formic acid in milliQ water, and buffer B was composed of 0.1 % formic acid in acetonitrile, with an elution gradient of 26-43% B between 0.21- 4 min, 80 % B between 4.01 - 5 min and then return to the initial conditions of 26% B at 5.01 min. The UV-detection was performed at 214 nm, and the concentration was determined using GLP-1 as external standard. The correct mass of the target peptide was confirmed by MS analysis. The MS data settings were set to 4 GHz at high resolution in a 100 to 3200 m / z mass range using dual ESI at positive mode and the spraying conditions were set to 325°C, using a N2 flow rate of 12 L / min, and the voltage set to 300V. The masses were deconvoluted using the Agilent MassHunter Qualitative Analysis B.07.00 program. The retention time is around 2.9 minutes and the target peptide molecular weight 4712 Da.

[0355] Dry Cell Weight Determination

[0356] A representative sample of the culture of 2 ml_ is pipetted into a pre-weighed centrifuge tube. The tube is then centrifuged at 4400 rpm for 5 min, after which the supernatant is discarded by decanting. The cell pellet is subsequently washed 2 ml_ saline, to rid the sample of medium components and the centrifuged, where after the wash is discarded by decanting. The wet biomass sample is dried at 105 °C Celsius for at least 24 hours and successively cooled down in a desiccator for 1 hour before weighing.

[0357] Data normalization

[0358] All values were normalized based on the average concentration of a high-yielding standard process, which was set as 100%. Each process' target peptide concentration and dry cell weight was then expressed as a percentage of this standard (relative target peptide concentration and relative dry cell weight). This allowed us to maintain the relative proportions and relationships between data points without revealing the actual numbers.

[0359] Example 1 - Target peptide related viscosity

[0360] To examine the high viscosity problem seen in the fermentation broth, an extensive rheological characterisation was performed on fermentation broth, supernatant and resuspended cells using strains with different cultivation pH.

[0361] Example 1.1 : Relationship between target peptide aggregation and viscosity.

[0362] An S. cerevisiae strain (yNN2071) was cultivated according to cultivation method 1 and samples were taken at specific time intervals to recover the target peptide by 2 different extraction methods (dilution method into water and alkalic extraction), and to characterise the rheological properties. The cultivation pH setpoint was set to 5.3 and kept constant. The results are shown in Table 1 and Fig. 1. Fig. 1 shows that the target peptide cannot be fully recovered by the dilution method and alkalic conditions are needed to further recover the target peptide. As can be seen in Fig. 1 and Table 1, the target peptide recovery by the dilution method is decreasing over cultivation time.

[0363] Table 1. Relative target peptide concentration (%) for samples from yNN2071 extracted by two methods (dilution and alkali).

[0364] This behaviour suggests that the target peptide has a strong tendency to aggregate. This was confirmed by increasing the pH of the sample such that the target peptide de-aggregated and could be detected by UPLC. Figs. 2A and B show a rheogram of a typical high viscosity cultivation sample. Fig. 2A shows a rheogram of a cultivation sample where the viscosity at various shear rates was analysed for the intact broth, its supernatant, and its pellet which was washed twice and resuspended in water. The results clearly show that the viscosity comes from the supernatant (and not the pellet). In addition, the viscosity gets lower with increasing shear rates which point to a pseudoplastic nature. Pseudoplastic viscosity has also thixotropic behaviour as prolonged shear exposure reduces the viscosity (see Fig 2B).

[0365] Fig. 3 shows that when the calculated amount of target peptide being aggregated (Caikaiic-Cdiiution) is plotted against the flow behavioural index (n), there is a strong negative linear correlation which shows that the more target peptide being aggregated the higher the pseudoplasticity (and thus viscosity) of the cultivation broth. Therefore, there is a strong indication that the target peptide is the actual cause behind the high viscosity that it is observed.

[0366] Conclusion:

[0367] As the cause of elevated viscosity / pseudoplasticity is found in the supernatant and is correlated to the aggregated peptide concentration, the aggregation of the product must occur in the soluble fraction.

[0368] Example 1.2: Confirming the link between broth pH, target peptide aggregation and viscosity

[0369] An S. cerevisiae strain (yNN2104) that tolerates a cultivation pH ranging from pH 4.5 to 5.5 for efficient recombinant production was cultivated according to cultivation method 1 to further investigate the relationship between viscous pseudoplastic broths and target peptide aggregation and also to determine the effect on primary recovery.

[0370] The cultivation was conducted at pH 5.3 and the fermentation broth was harvested and stored at 4 °C until analysis. The pH of the harvest was gradually increased, and broth samples with pH from 5.5 to 12.1 were taken, Theologically characterised and the concentration of the target peptide was measured by the dilution method as described above.

[0371] The results are shown in Table 2 and Fig. 4.

[0372] Table 2. Correlation between fermentation broth pH, relative target peptide concentration, viscosity at 2000 s-1, flow behaviour index n and flow consistency index k

[0373] As the broth pH increases, the more target peptide is detected by UPLC, the flow consistency index and viscosity are both reduced and the flow behaviour index increases (meaning less pseudoplastic and more Newtonian behaviour) (see Fig. 4). As can be seen in Fig. 4, a broth pH of around 8 is sufficient to de-aggregate the target peptide.

[0374] Conclusion:

[0375] A strong correlation between broth pseudoplastic viscosity, and target peptide aggregation is confirmed. In addition, the required pH to de-aggregate the target peptide is established to be around 8.

[0376] Example 1.3: Simplifying the recovery process and confirming the soluble state of the aggregated product

[0377] The benefits of alkalic treatment of the broth have been shown (Ex 1.1-1.2). However, strong alkalic treatment of the broth can lead to cell lyses and therefore release of host cell proteins (HCPs) and other unwanted contaminants. As the peptide aggregation occurs in the soluble fraction (see Ex 1.1 and 1.2), it was hypothesized that the alkalic treatment to de-aggregate the product does not need to take place in the broth but can be done on the supernatant instead.

[0378] Three different strains (yNN2071, and the other lineage strains yNN2101 and yNN2104 which show better growth and expression at higher pH levels) were cultivated according to cultivation method 1 (Table 3). yNN2071 was cultivated at pH 5.3 while yNN2101 and yNN2104 were each cultivated at pH 6.2. The harvest from each of the cultivations were obtained, clarified by centrifugation and the resultant supernatants were collected. In the supernatants, the concentration of the target peptide was measured a) without any treatment, and b) with alkalic treatment as described above.

[0379] Table 3. Relative target peptide concentration of broth supernatant of yNN2071 cultivated at pH 5.3, yNN2101 and yNN2104 cultivated at pH 6.2 extracted by centrifugation. The supernatant was then either analysed untreated or undergone an alkalic treatment (the same as the one described in material & methods as alkalic extraction). Untreated Alkalic treatment

[0380] As can be seen in Fig. 5, the target peptide concentration was very low in the cultivation run for strain yNN2071 with pH setpoint of 5.3 while for strains yNN2101 and yNN2104, which were cultivated with pH setpoint of 6.2, the target peptide concentration was significantly higher. However, when the pH of the supernatant was raised by the alkalic treatment, the concentration of the target peptide produced by strain yNN2071 was increased dramatically due to the target peptide getting deaggregated. On the other hand, in the case of the cultivation of strain yNN2101 and yNN2104 which were already cultivated at a high pH setpoint, the alkalic treatment only marginally increased the concentration of the target peptide measured by UPLC, therefore showing that the target peptide was already in the non-aggregated form. By avoiding applying harsh alkalic treatment on the fermentation broth but rather at the supernatant, we avoid cell lysis and increased release of impurities such as host cell proteins.

[0381] Conclusion:

[0382] This example confirms the soluble nature of the aggregated target peptide. It also shows that the cultivation pH is crucial for the formation of aggregates with lower cultivation pHs increasing the chances of formation of aggregates. In addition, it shows that in case the target peptide is aggregated during cultivation, there is no need to de-aggregate it before the clarification step, but it can be done after which will avoid unwanted cell lysis and thus avoid the increase of impurities by the action of the alkalic treatment.

[0383] Example 2 - Cell-derived viscosity

[0384] As shown in Example 1 , it is imperative to increase the cultivation pH setpoint to above 6.2 ideally to around 6.5 to avoid the formation of target peptide aggregates. To achieve this, new generation of strains were used which can grow and express recombinant products more efficiently at higher pH.

[0385] Example 2.1

[0386] The purpose of this experiment was to evaluate the expression and rheological properties of the fermentation broth when using the new lineage strains which can be cultivated at higher pH values. In Figs. 6A and B the cultivation of a new strain yNN2104 at pH 6.2 can be seen. Fig. 6B shows the dissolved oxygen tension, oxygen uptake rate and viscosity against cultivation time. Broth samples were taken at set time points and were processed using either the dilution method or the alkalic treatment before the concentration of target peptide was determined. The results are shown in Table 4 and Fig. 6A.

[0387] Table 4. The relative target peptide concentration from a pH 6.2 cultivation of the new lineage strain (yNN2104) as measured by the dilution method and alkalic extraction method to de-aggregate the target peptide.

[0388] Relative Target Peptide concentration (%)

[0389] Sample Cultivation time (h) Dilution method Alkalic, extr.

[0390] 1 95.6 83.1 80.4

[0391] 2 122.7 85.2 82.3

[0392] 3 143.9 90.4 84.2

[0393] 4 167.5 89.3 83.7

[0394] 5 218.7 80.0 81.0

[0395] 6 239.7 80.4 81.4

[0396] 7 263.4 83.5 79.7

[0397] 8 287.3 72.5 69.0

[0398] 9 311.7 60.7 58.6

[0399] 10 336.0 52.4 51.3

[0400] 11 407.6 39.7 38.1

[0401] 12 430.9 39.8 38.0

[0402] The target peptide concentration is comparable whether the dilution method or the alkalic extraction method was used. This means that there is no product aggregation. However, after 300 h, the dissolved oxygen tension of the culture is reduced, which is not due to increased oxygen uptake rate (OUR), but due to an increase in viscosity as can be seen in Fig. 6B. When broth samples were analysed, it was found that the cause of the viscosity is not in the supernatant - as was the case in Example 1 (target peptide related viscosity) but in the cell pellet (Fig. 7A).

[0403] In Fig. 7A, it can be seen that the viscosity is not of pseudoplastic nature but rather of shear thickening (dilatant) nature. In addition, the cultivation with a strain not expressing the target peptide showed exactly the same trend as shown in Fig. 7B. Hence, it can be concluded that the pellet is responsible for this type of viscosity.

[0404] Microscopic and macroscopic observations showed that the cells have the tendency to form clumps and growth on the wall and headplate of the bioreactors. Conclusion:

[0405] A new type of viscosity has been identified. In contrast to the product-related viscosity, this type of viscosity is coming from the pellet, presumably from the cells due to cell clumping, and it is of shear thickening behaviour. From now on, this type of viscosity will be referred as cell-derived viscosity.

[0406] Example 2.2 - Investigation of the relationship between viscosity and flocculation by the addition of Ca2+

[0407] The purpose of this experiment was to identify the root cause of the cell-derived viscosity. To that end, the clumping behaviour of the cells was considered. Additionally, it was assessed whether the cell-derived viscosity could be a result of flocculation.

[0408] The effect of cell flocculation by Ca2+on the rheological properties of a cell suspension is shown in Fig. 8. A sample from a high viscosity broth of strain yNN2201 was taken and aliquoted into 3 portions. First, the effect of cell flocculation was investigated by rheological characterizing: a) double-washed and resuspended pellet in de-ionized water, b) double-washed and resuspended pellet in a 0.02M EDTA solution, c) double-washed and resuspended pellet in a 25% (w / v) CaCl2 solution.

[0409] Calcium ions enable the cell surface flocculins to achieve their active conformation and bind the mannans in neighbouring cells (Soares EV, J Appl Microbiol. 2011 Jan;110(1):1-18. doi: 10.1111 / j.1365-2672.2010.04897.x. Epub 2010 Nov 29. PMID: 21114594). The rheogram of Fig. 8 shows that the addition of calcium dramatically reduces viscosity and flattens its rheogram, which points to a Newtonian fluid behaviour. On the other hand, sequestering calcium by the addition of EDTA, results in an even higher viscosity level and significantly more shear thickening behaviour at shear rates higher than 500 s-1(critical shear rate). Therefore, Ca2+seems to have a significant effect on reducing broth viscosity by increasing cell flocculation and therefore, particle compactness (Bliatsiou et al., Biochem. Eng. J. 2020; 163, 107746).

[0410] To build upon the previous findings and to provide potential solution for the cell-derived viscosity, Ca2+was added during continuous cultivation after the increase of the cell-derived viscosity.

[0411] The addition of Ca2+during cultivation of the new generation of hosts (Fig. 9 at 409 h cultivation time) causes a significant decrease in the flow behavioural index (n) of the broth signifying an increase in the pseudoplasticity. The viscosity when measured at high shear rates was dropped while the flow consistency index (k) increased. This clearly show that the addition of Ca2+results in a decrease in shear thickening type of viscosity coming from the cells and an increase in the shear thinning behaviour. The question of where the shear thinning viscosity comes from can be answered by looking at the relative target peptide concentration extracted by the dilution method and alkalic treatment before and after the addition of Ca2+in Fig. 9E. More specifically, the addition of calcium causes the target peptide to aggregate. This finding is in line with previous works on aggregation / fibrillation prone proteins and peptides showing that Ca2+and high ionic strength significantly increases aggregation and fibrillation (Urrutia et al., FEBS Lett. 1989 Apr 10;247(1): 17- 21. doi: 10.1016 / 0014-5793(89)81230-x. PMID: 2707446; Huang et al, Biochem Biophys Res Commun. 2022 May 7;603:13-20. doi: 10.1016 / j.bbrc.2022.02.097. Epub 2022 Feb 25. PMID: 35276458). The property of Ca2+to cause target peptide aggregation and an increase in broth pseudoplastic behaviour can also be supported by the behaviour of the dummy strain yNN1953 (not expressing the target peptide). For yNN1953, the addition of calcium has little effect on the flow behaviour index (Fig. 9D) or on the flow consistency index (Fig. 9B), but has significant effect on high shear viscosity, which is reduced as demonstrated in Fig. 9A. Since there is no target peptide to get aggregated by the presence of Ca2+and to cause an increase in viscosity and pseudoplasticity, the cell-derived viscosity is reduced due to flocculation and this contributes to an overall reduction in broth viscosity.

[0412] Conclusion:

[0413] In view of the above, it can be concluded that flocculation is not the cause of the cell-derived viscosity. Conversely, it appears to help eliminating this type of viscosity on fermentation samples. However, since the addition of Ca2+, used to flocculate the culture, also causes the aggregation of the target peptide, it is not suitable to add Ca2+to avoid cell-derived viscosity, because it would merely lead to an increase in the pseudoplastic type of viscosity.

[0414] Example 2.3. FLO11 activity and viscosity

[0415] The purpose of this experiment was to investigate whether the observed cell clumping and presumably the high shear thickening viscosity is the result of the activity of the FLO11 gene which is responsible for the biofilm formation in yeast.

[0416] To that end, strains with knockouts in FLO11 and FLO8 genes were produced and tested in long continuous cultivations under standard conditions with pH setpoint at 6.5 to eliminate the effect of the viscosity coming from the product. FLO8 is involved in the transcriptional activation of FLO11 which in turn is involved in biofilm formation. Fig. 10 shows the viscosity profiles of the FLO8 and FLO11 knockout strains with the wild type as control. As can be seen, there is little to no improvement in the broth viscosity of the knockoutstrain cultivations.

[0417] Conclusion:

[0418] Biofilm formation via the action of the FLO8 and FLO11 gene is not the cause of the shear thickening viscosity observed in the fermentation broths of the yNN2104 derived strains.

[0419] Example 2.4 - Effect of AMN1 activity on broth viscosity

[0420] The purpose of the experiment was to investigate the potential link between the shear thickening viscosity and the AMN1 activity. Another potential cause of the clumping behaviour and potentially the viscosity may be the incomplete mother to daughter cell separation. In this case, the cells form small fluffy clumps which can potentially increase viscosity. AMN1 is a gene involved in post-mitotic cell separation and it has been shown that the A / W / V13680allele is the cause of the incomplete separation of mother to daughter cell (Fang et al., PLoS Genet. 2018 Oct 1 ;14(10):e1007691. doi: 10.1371 / journal.pgen.1007691. PMID: 30273335; PMCID: PMC6181423). The strain variants of the new host cell lineage including yNN2104 and yNN2993 indeed harbour the AM N680allele and for that reason an AAMN1 (yNN2993) version of the strain was prepared to be tested.

[0421] Results:

[0422] Fig. 11 shows that by deletion of the AMN1 gene (strain yNN2993), not only the cell clumping phenotype was eliminated (Fig. 11 E) but also the viscosity dropped to very low levels and the pronounced shear thickening behaviour after 300h disappeared (Fig. 11A and B). The deletion of the AMN1 gene eliminated the viscosity and therefore allowed for cultivations with no oxygen mass transfer challenges resulting in the dissolved oxygen tension being maintained at adequate levels throughout the long cultivation (Fig. 11C).

[0423] The connection between the cell clumping behaviour and high viscosity broths can be linked to the nature of the clumps. In the case of the A / W / Vf-triggered clumps, the clumps are relatively small and fluffy with irregularities in surface*** all of which lead to suspensions with higher viscosity. On the other hand, in the case of Ca2+induced flocculation as described in Example 2.2, the clumps are bigger and more compact which makes the suspension viscosity significantly lower.

[0424] *** The higher viscosity resulting from irregular particle surfaces can be attributed to two factors. Firstly, the flow lines of the solvent flowing around these particles are more strongly deflected than for same-size spheres. Secondly, the probability of particle-particle interactions increases due to the greater specific surface area of the irregular particles. This effect is particularly pronounced at high particle concentrations and high shear rates.

[0425] The cell wall of yeast is composed of two layers: an inner skeletal layer made up of chitin and betaglucan, which provides structural support and maintains cell shape, rigidity, and turgor pressure, and an outer layer of glycosylated mannoproteins, which are involved in specific cell functions such as adhesion and invasion. During cell division, when mother and daughter cells are about to separate, the activity of glucanases and chitinases is required to break down the cell wall and allow for separation to occur (Hall et al., Cell Surf. 2022 Jan 12;8:100074. doi: 10.1016 / j.tcsw.2O22.100074. PMID: 35097244; PMCID: PMC8783092). The AMN1 gene has been shown to play a role in regulating the activity of glucanases and chitinases during yeast cell division (Koschwanez et al., Elife. 2013 Apr 2;2:e00367. doi: 1O.7554 / el_ife.OO367. PMID: 23577233; PMCID: PMC3614033). When cell division is hampered, glucanase and chitinase expression are inhibited to prevent premature cell-daughter separation. In the presence of the AMN368Dallele variation expression of these enzymes may possibly be inhibited (Koschwanez et al., 2013).

[0426] Conclusion:

[0427] The AMN1 activity is responsible for the cell clumping behaviour of the cells and confirms the hypothesis that the cell clumps cause the shear thickening broth viscosity. Removal of the gene leads to complete elimination of the viscosity problem and to a significant increase in the dissolved oxygen tension levels in the fermentation broth.

[0428] Example 2.5. Further investigation of the role of AMA / 1 . The effect of gene deletion versus replacement of the AMN clumping allele with a non-clumping ZWA / 1D368Vallele on broth viscosity and fermentation performance

[0429] The purpose of this experiment is to determine whether the elimination of the viscosity when the AMN1 gene is deleted is due to the removal of the clumping behaviour of the cells. Put differently, this experiment was set up to confirm that the shear thickening cell-derived broth viscosity is due to the clumping behaviour derived from the AMN1 gene and exclude the possibility that the viscosity is related to some other function of the AMN1 gene product.

[0430] Therefore, to confirm the link between cell-derived shear thickening viscosity and the clumping behaviour of the cells caused by AMN1 , a non-clumping version of the AMN1 gene (D368V) was used to replace the clumping AMN allele. The cultivations were run at cultivation pH of 6.2, temperature of 29 °C and dilution rate of 0.08 IT1.

[0431] Results

[0432] Fig. 12A shows the dissolved oxygen tension during a 600 h cultivation of the AMN1 wild type strain (yNN2104), the AAMN strain (yNN2993) and the strain with non-clumping AMN1 allele (AMN1D368Vfrom now on, strain: yNN3073). It is evident that the yNN2104 strain carrying AMN1 wildtype could not maintain a stable dissolved oxygen tension after 220h cultivation time. The reason behind the lower dissolved oxygen levels in the wild type is not due to elevated oxygen uptake rate after 220h (it is identical for all culture Fig. 12. B) neither due to the higher biomass (Fig. 12C) but due to higher viscosity (Fig.12D, viscosity as measured at a shear rate similar to the one existing in the bulk medium of the bioreactor). Fig. 12E shows that the flow behaviour index is significantly higher for the wild type strain (yNN2104) culture and therefore more dilatant than the other two cultures.

[0433] It is worth mentioning that since the cultivation pH was 6.2, there is some limited amount of target peptide being aggregated, causing pseudoplastic viscosity, which may cause a reduction in the flow behaviour index.

[0434] Finally, Fig. 12F shows that the deletion of AMN1 or replacement of the AMN1 gene with a nonclumping AMN1D368Vallele does not have a negative implication on the expression of the target peptide.

[0435] Conclusion

[0436] The shear thickening cell-derived viscosity is the result of the clumping activity caused by the AMN wild type gene and either gene deletion or replacing the AMN1 gene with a non-clumping AMN1D368Vallele can remove the viscosity without negatively affecting other physiological characteristics of the cultures such as the oxygen uptake rate, biomass formation or expression of the target product.

[0437] Example 2.6. Confirming the effect of cultivation pH on product-related viscosity in a cultivation of the strain with the cell clumping phenotype removed (AAMN1}

[0438] This experiment was run to confirm the effect of pH on product-related viscosity but this time, the target peptide was expressed by the new generation of strains where the AMN1 gene was deleted to avoid interference in the rheological characterization by the cell-derived viscosity. The cultivation pH is reduced from 6.4 to 5.3. Fig. 13 clearly shows that as soon as the cultivation pH drops to 5.3, the viscosity increases immediately, and the flow behavioural index drops which means that the pseudoplasticity increases. As expected, the ratio of target peptide measured by the alkalic extraction over the concentration of target peptide measure by the dilution method (from now on this ratio is called aggregational index) correlates very well with the increase of both viscosity and pseudoplasticity proving that the cultivation pH is determining the extent of target peptide aggregation and viscosity and that high cultivation pHs completely eliminate the product-related viscosity.

[0439] Conclusion:

[0440] This experiment clearly shows that the pseudoplastic behaviour deriving from the supernatant is independent of the expressing strain and confirms the previous observations that it is product- related and can be triggered at low cultivation pH.

[0441] Example 2.7. Confirming the effect o AMN1 on the cultivation performance of the yNN0381 strain (derived from the model strain EM93)

[0442] The purpose of this experiment was to confirm the effect of the clumping AMN 7368Dallele on the viscosity profile of the continuous culture of a publicly available strain. For this reason, a strain derived from EM93 (yNN0381) was chosen as this strain harbours the AMN1368Dallele responsible for the cell clumping phenotype.

[0443] Continuous cultivations of the AMN~\ wild type strain (yNN0381), the AA / WA / 1 strain (yNN3497) and the AMN1D368Vmodified strain (yNN3521) were performed at cultivation pH 6.2, temperature 28 °C, dilution rate 0.08 IT1.

[0444] Fig. 14 shows that the viscosity of the AAMN1 and AMN1D388Vstrains (yNN3497 and yNN3521) is minimal compared to the wild type AMN1 (yNN0381), accompanied by a reduction of cell clumping when observed under the microscope, confirming that the clumping allele of AMN1 is responsible for the shear thickening cell-derived viscosity. Therefore, by gene deletion or by introducing the nonclumping version of the AMN1 gene, we can completely avoid cell clumping and have very low broth viscosities.

[0445] Conclusion: These results confirm that the 368D allele of the AMN1 gene is not only responsible for cell clumping but also for the increased cell-derived viscosity. Deletion of the AMN1 gene or exchanging the genomic version with the non-clumping AMN1D368Vallele eliminates the viscosity problem.

[0446] Example 3. Eliminating both product-related and cell-derived viscosity leads to cultivation broths with excellent rheological properties

[0447] This example does not contain any new experiments but summarises the previous findings.

[0448] Fig. 15 shows the viscosity at shear rate of 250 s_1and flow behaviour index of a typical fermentation with the old (acidophilic) generation of strains where the cultivation pH is 5.3 leading to highly viscous, very pseudoplastic broths (n « 1). This highly pseudoplastic behaviour is especially problematic in large industrial scale bioreactors, where large shear gradients exist, e.g., very high shear rate close to impellers and very low shear rate as we move away from the agitator. In the very low shear rate areas, the viscosity will be very high which will aggravate mass and oxygen mixing and will result in cells being starved of oxygen thus going into fermentative metabolism producing energy-wasteful and deleterious by-products such as ethanol and acetic acid and eventually lowering the product yield.

[0449] On the other hand, the new generation of strains with the wild type AMN1368Dgene cultivated at pH 6.2, may not have significant viscosity coming from the product aggregation, however, the clumping phenotype results in high viscosity shear thickening broths (n »1) which also contribute to exacerbated gradients but, in this case, the high viscosity areas and therefore low mass and oxygen mixing areas will be in close vicinity to the bioreactor’s impellers. The suggested process, i.e. the use of strains with AMN1 gene deletion or AMN1D368Vmutation cultivated at high pH to avoid the target peptide aggregation, results in cultivation broth with very low viscosity and completely Newtonian behaviour (n = 1) and thus the oxygen and mass gradients in general are alleviated allowing for high product yields.

[0450] Conclusion:

[0451] Expressing the target peptide in strains without the cell clumping phenotype of the AMN1 gene carrying the 368V allele at high cultivation pH (pH > 6.4) results in fermentation broths with minimal viscosity of completely Newtonian behaviour allowing for more efficient processes, that is, reduce the energy consumption and cooling requirements and increase the product yields.

[0452] Example 4 This example aims to verify that the phenomena of product related viscosity and cell derived viscosity also hold true when [Arg34]GLP- 1(9-37) is fused to other extensions and that these viscosities can be overcome by high cultivation pH and genetic manipulation to circumvent the clumping AMN1 activity respectively.

[0453] Example 4.1: Cultivation broth viscosity of strains expressing fArq341GLP-1(9-37) with different extensipns

[0454] This experiment tests the broth viscpsity of strains with and without the clumping AMN1 gene expressing [Arg34]GLP- 1 (9-37) fused to extension 3 or 4 instead of extension 1 used in previous Examples. The cultivations were initially run at pH 6.5 to avoid “product-related” viscosity due to product aggregation. Subsequently, the cultivation pH was gradually reduced in stages until it reached 5.3 as shown in Fig. 16.A (all other conditions were set as described before). The drop in pH was carried out in order to investigate the extend of product aggregation and how it affects the “product-related” viscosity when [Arg34]GLP-1 (9-37) is fused to these different extensions.

[0455] Results

[0456] The [Arg34]GLP-1(9-37) concentration levels obtained in the AMN1 knock-out strains for both extensions are equal to or higher than those achieved with the same target peptides in the wild type AMN1 strains (Fig. 16. B). Fig. 16. C. shows only minor to negligible product aggregation during cultivation at pH 6.5. This shows that cultivation at pH 6.5 is sufficient to keep the aggregation levels low as we observed for [Arg34]GLP-1(9-37) with extension 1 in cultivations at pH 6.2. The viscosity profile is shown in Fig. 16. Du where the viscosity was measured at a shear rate of 2000s'1. The AMN1 knockout strains have consistently lower viscosities than the respective AMN1 wild type strain bearing the AMN1 clumping D368 allele, although, the difference is not as high as observed for strains expressing [Arg34]GLP- 1(9-37) with extension 1 cultivated at pH 6.2. The flow behaviour index (Fig. 16. Eu) is also lower for the AMN1 knockout strains as expected as the more viscous broths of the AMN1D368strains have a more shear thickening behaviour (n > 1) than the cultivation broths from the AMN1 knockout strains.

[0457] After 392h of cultivation, the pH was gradually reduced until it reached 5.3 as can be seen in Fig. 16. A. This pH drop caused product aggregation as can be inferred by subtracting the two extraction methods (alkalic extraction - dilution method, see General Methods of Detection and Characterisation). The aggregations seemed to be higher in the cultivation of strains yNN7092 AAMN1) and yNN7095 (AMN1 wildtype) expressing [Arg34]GLP-1 (9-37) with extension 3 (Fig. 16. C). As expected, the increased product aggregation resulted in a significant increase in the broth viscosity of all the cultivations regardless of the AMN1 status. The flow behaviour index (Fig. 16. Ej), indicates that [Arg34]GLP-1 (9-37) aggregation caused the broth to become more pseudoplastic (n < 1). These observations are similar, albeit to a lesser extent, to what was previously observed with strains expressing [Arg34]GLP-1(9-37) with extension 1 (increased broth viscosity of pseudoplastic nature coming from the liquid fraction of the broth). It is worth noting that the pseudoplasticity of the broth was more apparent in the case of the AMN1 knockout strains, as the AMN1 wild type cell clumping confers shear thickening properties which to an extent counterbalance the pseudoplasticity caused by the aggregated target peptide.

[0458] Conclusions:

[0459] The knockout of the AMN1 gene causes a significant and consistent decrease in the culture broth viscosity of the strains expressing [Arg34]GLP-1(9-37) with extension 3 and 4. Similar to the results from the previous examples of [Arg34]GLP-1(9-37) with extension 1 to avoid the product-related viscosity due to the target peptide being aggregated, cultivation at a pH setpoint of > 6.2 is sufficient to eliminate this type of viscosity. Therefore, to ensure a Newtonian low viscosity cultivation broth, the use of an AMN1 knockout strain cultivated at a pH of higher than 6.2 is sufficient regardless of the extension [Arg34]GLP-1 (9-37) is fused to.

[0460] Example 4.2

[0461] At the end of the cultivation of strain yNN7093 (AAMNT) expressing [Arg34]GLP- 1 (9-37) with extension 4, the cultivation broth was subjected to a gradual increase in pH using NaOH, while samples were taken, and the supernatant was analysed. Fig. 17. A shows the viscosity measurement of the supernatants derived from broth at different pH values plotted against shear rate. There is an obvious flattening of the curve and a reduction of viscosity at lower shear rates as the pH increases. Fig. 17. B. shows the correlation between fermentation pH and peptide aggregation and consequently the effect on the rheology of the resulting broth supernatant in the case of [Arg34]GLP-1(9-37) with extension 3. This was performed in a similar fashion to Fig. 4, that shows results for [Arg34]GLP- 1(9-37) with extension 1. As the broth pH increases, the target peptide becomes de-aggregated (aggregated precursor concentration decreases), the flow consistency index and the viscosity at 2000s'1decreases, and flow behaviour index increases, approaching the unity, signifying less pseudoplastic and more Newtonian behaviour. The rheological characterization was performed in the broth supernatant, which indicates that the target peptide, when aggregated, stays in the soluble fraction of the broth as was observed for the [Arg34]GLP-1 (9-37) fused to extension 1

[0462] Conclusions: This experiment confirms the effect of pH on aggregation of [Arg34]GLP-1 (9-37) , when fused to different extensions (extension 3 and 4). In short, as we also observed for strains expressing [Arg34]GLP- 1(9-37) fused to extension 1 , [Arg34]GLP-1(9-37) fused to extension 3 or 4, when aggregated at low cultivation pH, causes high viscosity of pseudoplastic nature while remaining in the soluble fraction of the cultivation broth. To de-aggregate the already aggregated target peptide, increasing the broth pH to above 8 is sufficient. Most importantly, this treatment can be performed after the clarification step, as the aggregated target peptide is still in the soluble fraction and avoid contamination with intracellular material caused by cell lysis.

[0463] Example 5

[0464] This example aims to verify that the viscosity caused by AMN1 related clumping of the cells is independent of the recombinant peptide or protein expressed. In this experiment, four GLP1-GIP fusion formats were expressed in S.cerevisiae strains harboring the clumping AMN1D368gene or having a deletion of the AMN1 gene.

[0465] Example 5.1: Cultivation of other GLP-1 related formats and confirmation of the A / W / V7-derived viscosity

[0466] This work aims to confirm once again the effect of AMN1 activity on the rheological characteristics of the resulting broths of S.cerevisiae strains. In addition, the effect of cultivation pH of 6.2 on the target product aggregation and the rheological behaviour of GLP1-GIP fusion precursor peptides is assessed.

[0467] Results

[0468] There is an obvious oxygen limitation in all strains carrying the clumping AMN1 gene (D368 allele) regardless of the target peptide being expressed (Fig. 18 Ai-Di). This occurs after around 320 to 350h of cultivation time. At this timeframe, there is no increase in the oxygen demand of the cultures as the oxygen uptake rates remain stable throughout the process (Fig. 18 Aii-Dii). Likewise, the biomass levels remain relatively stable and on par with the AMN1 knock-out (AAMN1) strains. Therefore, the reason behind the oxygen limitation in the clumping AMN1 strains is the reduced oxygen transfer due to the higher viscosity levels (Fig. 19Ai-Di). All strains expressing the four different GLP1-GIP fusion precursor peptides had almost identical viscosity profiles. The AAMN1 strains had consistently lower viscosities which fluctuated between 2.5-2.8 cP at shear rate of 2000 S’1, whereas the clumping AMN1 strains had viscosities higher than 3.2 cP at shear rate of 2000 S’1. The clumping AMN1 wild type strains showed a further increase in viscosity after 320h cultivation time as we have observed in the previous examples with strains expressing other GLP1 formats. The average viscosity values at shear rate of 2000s’1before and after the viscosity increase (at ~320h) are presented in bar plots of Fig. 20. The error bars of Fig. 20, represent the variability of the viscosity values within the specified time intervals. This clearly highlights the significance of the differences in the viscosity values between AMN1 wild type and AAMN1 strains, both before and especially after the 320h timepoint.

[0469] Fig. 19.Aii-Dii. shows that deletion of the AMN1 gene has no negative effect on the expression of the GLP1-GIP fusion precursor peptides as the resulting peptide concentrations are identical between the AAMN1 strains and AMN1 wild type strains. Specifically, for GLP1-GIP fusion precursor 1 with extension 5 (ext5-GLP1GIP-1) the target peptide concentration is 55.6 % (AMN1 wild type) and 57.9 % (AAMN1), for GLP1-GIP fusion precursor 1 with extension 6 (ext6-GLP1GIP- 1) the target peptide concentration is 58.6 % (AMN1 wild type) and 58.1 % (AAMN1), for GLP1-GIP fusion precursor 1 without extension (GLP1GIP-1) the target concentration is 36.9 % (AMN1 wild type) and 35.2 %( AMN1), for GLP1-GIP fusion precursor 2 without extension (GLP1GIP-2) the target concentration is 25.5% (AMN1 wild type) and 25.9 % (AAMN1). In all cases the target peptide concentration is calculated by integrating along the given time intervals using the composite trapezoidal rule and dividing by the specific time intervals. Fig. 19.Aiii-Diii shows a low level of target peptide aggregation in all the cultivation which do not exceed the 5% of relative peptide concentration. This shows that a cultivation pH of 6.2 does not completely eliminate peptide aggregation and higher cultivation pH should be pursued if it is to completely eliminate the phenomenon. This is similar to the case of [Arg34]GLP-1(9-37) (SEQ ID No 2), where the cultivation pH should be above 6.4 to completely eliminate peptide aggregation and thus product-related viscosity. In the current experiment, the low aggregation levels do not seem to significantly influence the viscosity of the broth.

[0470] Conclusions:

[0471] There is a clear difference in the broth viscosity between the clumping AMN1 strains and AMN1 knock-out strains expressing the four different GLP1-GIP fusion peptides. The difference between the two host strains (AAMN1 and AMN1 wild type) becomes even more pronounced after 320h as we also observed for the [Arg34]GLP-1(9-37) cultivations (Examples 2.1 , 2.3, 2.4). The deletion of the AMN1 gene offers a substantial process improvement as the clumping AMN1D368wild type cultures face low dissolved oxygen tension after 320h due to the high viscosity (the oxygen consumption is the same for all strains) which impairs the oxygen transfer in the fermentation broth. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS1. A variant Saccharomyces cerevisiae cell, wherein the variant cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant cell has been modified such that the D368 allele has been knocked out or has been modified to a D368V allele; and wherein the variant cell further comprises a polynucleotide vector encoding a target peptide, wherein the target peptide is a GLP1-GIP polypeptide or an enterokinase-cleavable fusion polypeptide comprising as polypeptide of formula (I):Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 whereinZ2is an optional polypeptide or amino acid residue;X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;X5is selected from the genetically encoded amino acids but S and I;Xe is absent or selected from the genetically encoded amino acids; andZ1 is selected from the group consisting of K34R-GLP-1(7-37), K34R-GLP-1(9- 37), EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS and EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS.

2. The variant Saccharomyces cerevisiae cell according to claim 1 , wherein X4 is E, Q, L, G, or A, preferably wherein at least one of X4and X5is E or D.

3. The variant Saccharomyces cerevisiae cell according to claim 1 or claim 2, wherein X5-X4 is selected from the group consisting of DE, DD, DL, DQ, DG, DA, DS, EE, EQ, EL, ED, EG, EA, ES, QE, HE, NE, and ME.

4. The variant Saccharomyces cerevisiae cell according to any one of claims 1 to 3, wherein X5-X4 is selected from the group consisting of DD, DE, DL, DQ, EE, and EQ.

5. The variant yeast cell according to any one of claims 1 to 4, wherein X5-X4 is DD or DE.

6. The variant Saccharomyces cerevisiae cell according to claim 1, wherein the GLP1-GIP polypeptide is EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 4) or EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5).

7. The variant Saccharomyces cerevisiae cell according to any one of claims 1 to 6, wherein the variant Saccharomyces cerevisiae cell is capable of expressing a target peptide in higher yields at apH above 5.5, such as at a pH in a range of about 5.5-12, than at a pH below 5.5, such as at a pH in a range of 5.4-1.

8. Use of a variant Saccharomyces cerevisiae cell for the recombinant production of a GLP-1 peptide or GLP1-GIP polypeptide, wherein the variant Saccharomyces cerevisiae cell comprises an AMN1 gene and wherein the D368 allele of the AMN1 gene has been modified such that the D368 allele has been knocked out.

9. Use of a variant Saccharomyces cerevisiae yeast cell for the recombinant production of a GLP-1 peptide or GLP1-GIP polypeptide, wherein the variant Saccharomyces cerevisiae cell comprises an AMN1 gene and wherein the D368 allele of the AMN1 gene has been modified such that the D368 allele has been modified to a D368V allele.

10. The use according to claim 8 or claim 9, wherein the GLP-1 peptide is of formula (I):Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 whereinZ2is an optional polypeptide or amino acid residue; 4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;X5is selected from the genetically encoded amino acids but S and I;X6is absent or selected from the genetically encoded amino acids; andZ1 is K34R-GLP- 1(7-37) or K34R-GLP-1(9-37).

11. A method of increasing the expression of a peptide, the method comprising the steps of a) providing a variant Saccharomyces cerevisiae cell, wherein the variant Saccharomyces cerevisiae cell is derived from a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele, and wherein the variant yeast cell has been modified such that the D368 allele has been knocked out or has been modified to a D368V allele; b) transforming the variant Saccharomyces cerevisiae cell of step a) with a polynucleotide vector encoding a GLP1-GIP polypeptide or an enterokinase-cleavable fusion polypeptide of formula (I):Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 whereinZ2is an optional polypeptide or amino acid residue; 4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;Xs is selected from the genetically encoded amino acids but S and I;Xs is absent or selected from the genetically encoded amino acids; andZi is a GLP-1 peptide or a functional variant thereof; c) inoculating a fermentation medium with the transformed yeast cell obtained or obtainable in step b); d) adjusting the pH of the inoculated fermentation medium obtained or obtainable in step c) to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint, e) cultivating the inoculated fermentation medium obtained or obtainable in step d) at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the polypeptide in the fermentation broth, f) harvesting the fermentation broth, g) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation, h) recovering the polypeptide, and optionally i) purifying the polypeptide recovered in step g.

12. The method of according to claim 11 , wherein Zi is K34R-GLP 1(7-37) or K34R-GLP 1(9-37).

13. A method for producing a target peptide comprises the steps of a) providing a parental Saccharomyces cerevisiae cell comprising an AMN1 gene with a D368 allele; b) deleting the D368 allele or modifying the D368 allele to a D368V allele to obtain a variant Saccharomyces cerevisiae cell; c) transforming the variant Saccharomyces cerevisiae cell with a polynucleotide vector encoding a target peptide or a precursor thereof; d) inoculating a fermentation medium with the transformed variant Saccharomyces cerevisiae cell obtainable in step c; e) adjusting the pH of the inoculated fermentation medium obtainable in step d) to a desired pH setpoint, wherein the pH setpoint is in the range of above 5.5, and keeping the pH constant at said pH setpoint;f) cultivating the inoculated fermentation medium obtainable in step e) at a temperature between 20 °C and 35 °C, such as between 25 °C and 30 °C, to secrete the target peptide in the fermentation broth; g) harvesting the fermentation broth; h) clarifying the fermentation broth to obtain a soluble fraction, optionally by centrifugation; i) recovering the target peptide; and optionally h) purifying the target peptide recovered in step g.

14. The method according to claim 13, wherein the target peptide is a GLP1-GIP peptide or an enterokinase-cleavable fusion polypeptide of formula (I):Z2-X6-X5-X4-G-D-R-ZI (I) SEQ ID NO: 1 whereinZ2is an optional polypeptide or amino acid residue;X4 is E, Q, L, G, A, S, F, H, Y, W, T, or M;Xs is selected from the genetically encoded amino acids but S and I;X6is absent or selected from the genetically encoded amino acids; andZ1 is a GLP-1 peptide or a functional variant thereof; and optionally wherein the GLP-1 peptide is K34R-GLP- 1(7-37) or K34R-GLP-1(9-37).

15. The method of according to any one of claims 11-14, wherein the pH setpoint is in the range of 6.0-8.5, such as in the range of 6.1-8.5.

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