Biosensors for monitoring cellular stress

Abstract

The present invention relates to a biosensor nucleotide construct for monitoring of periplasmic protein or peptide production in a host cell, comprising an inducible promoter responding to a stress factor of the host cell, wherein the promoter is operably linked to a gene encoding a biomarker.

Description

Biosensors for monitoring cellular stress.Field

[0001] The present disclosure describes a genetic biosensor having an inducible promoter operably linked to a gene encoding a biomarker for monitoring the production of proteins or peptides in host cells. Also described herein are expression vectors and host cells hosting the genetic biosensor as well as the use of the genetic biosensor in methods for detecting cellular stress, for monitoring production of a protein of interest and / or for optimisting metabolic pathways of the host cells. Finally described herein is a kit of parts useful introducing the genetic biosensor not a host cell of interest and for perfoming the said methods.Background

[0002] Antibody fragments, protein hormones and other useful recombinant proteins are often produced in microorganisms, for example in the periplasm of Escherichia coli (see refs. 1 to 4). In particular some proteins are finally produced in the periplasm of host cells, because the periplasm contains enzymes required for the formation of disulphide bonds, which for some proteins are necessary for folding and function of these proteins (see refs. 5 to 7). Periplasmic production of proteins also offers the possibility to avoid cytoplasmic proteases, to control the N-terminal amino acid (following signal peptide cleavage), and to simplify downstream purification processes (see ref. 8).Periplasmically produced or finished recombinant proteins are trafficked to the periplasm by fusing the coding sequence to the coding sequence of an N-terminal signal peptide (see refs. 8 and 9). The signal peptide, together with the chaperones' Trigger factor and SecB, delays folding of the protein, so that it can be secreted through the Sec translocon (see refs. 10 to 12). Secretion is facilitated by SecA and other auxiliary modules, such as SecDFYajC and PpiD (see refs. 8 and 13). Once the recombinant protein is translocated to the periplasm, the signal peptide is cleaved by the signal peptidase LepB, and the protein folds with the assistance of a network of periplasmic chaperones disulphide catalysts and isomerases (see refs. 14 and 15).

[0003] This shows that production of recombinant proteins in the periplasm is a challenging endeavour. If the expression levels exceed the capacity of the Sec translocon, the recombinant protein will start to accumulate in the cytoplasm (see refs, (see refs. 4, 8, 15 to 17) resulting in a heat shock response, impaired energy metabolism, and reduced cell growth (i.e. biomass) (see refs. 17 to 19). The expression levels can also exceed the chaperone capacity in the periplasm, causing the recombinant protein to misfold and / or to aggregate (see ref. 8), causing an envelope stress response and potentiallycell death (see ref. 20). Maximising production titres for periplasmic proteins or other proteins causing a cellular stress response is therefore a tedious process of tuning expression conditions (inducer concentration, induction time, media, temperature) for maximal titres of secreted and soluble protein (see refs 4, 16, 21). Secretion and solubility is a widely accepted approximation for folded and functional protein (see ref. 22), which can be assessed using classical biochemical approaches or variants thereof, such as cell lysis followed by fractionation, SDS-PAGE and Western blotting (see ref. 23). However, these methods are not compatible with automated screening of single-cells eg. by Fluorescence-activated Cell Sorting (FACS) or other high throughput screening approaches. Accordingly, there is a need for improved tools and methods for measuring and monitoring production of recombinant protein in host cells.

[0004] Schlegel et al ; Microbial Cell Factories; Springer; vol. 12; no. 1; 12 March 2013 (2013-03-12); pages 12-24; xp021141763; ISSN: 1475-2859, DOI: 10.1186 / 1475-2859-12-24 pertains to expression and secretion of both SFGFP and the single-chain variable antibody fragment. The paper describes how the Sec-translocon capacity in E. coli can be saturated, but it does not relate to biosensors for monitoring cellular stress.

[0005] Kim et al.; Frontiers in Molecular Biosciences; vol. 8; page 1-14; 11 May 2021 (2021-05-11); xp93118172; ISSN: 2296-889x, DOI: 10.3389 / fmolb.2021.678697 relates periplasmic chaperones which counter acts stress in bacteria such as by preventing protein aggregation, disaggregating aggregated proteins, and aiding in protein folding. However, this paper does not pertain to stress sensitive promoters or genes encoding biomarkers suitable for stress detection or to the combination into a biosensor construct expressing the detectable biomarker in response to a stress.

[0006] Keller et al.; Biochemistry; vol. 54; no. 23; 1 June 2015 (2015-06-01); pages 3670-3676; xp93118161; ISSN: 0006-2960; DOI: 10.1021 / acs.biochem.5b00242 pertains to sensing of the properties of lipid bilayers in mutant strains lacking phosphatidylethanolamine. However, this paper does not relate to biosensors for monitoring stress caused by protein or peptide production in a cell, rather it describes sensing change in lipid properties caused lack of expression of phosphatidylserine synthase leading the cell to be deficient of phosphatidylethanolamine thereby changing the lipid bilayer.

[0007] Zoheir et al.; NPJ Biofilms and Microbiomes; vol. 9; no. 1; 21 august 2023 (2023-08-21); xp93118503; ISSN: 2055-5008, DOI: 10.1038 / s41522-023-00424-l pertains to biosensors which are not built or suitable for detection of stress caused by periplasmic production of proteins or peptides.

[0008] The production of recombinant proteins or peptides in high titres, in particular periplasmicproteins or peptides, is a challenging endeavour for those working in both academia and industry. It hinders the advancement of fundamental knowledge, as well as the commercialisation of biotechbased solutions and new therapeutic targets, and there is a need for providing further techical means and methods for monitoring production of compound of interest, such as proteins or peptides in host cells.Summary

[0009] Two major OFF-pathway events have been identified that need to be avoided in the production of particularly recombinant periplasmic compounds such as proteins or peptides: (1) inefficient secretion from the cytoplasm and (2) misfolding and aggregation in the periplasm (see refs 4 and 17). These OFF-pathways events cause cellular stresses, which initiates a program of transcriptional re-wiring that helps the cell regain homeostasis (see refs 18 and 20). Stress induced transcriptional reprogramming is detrimental to recombinant protein or peptide production as it affects the ability of the cell to sustain protein or peptide synthesis. It also affects cellular energy metabolism and cell growth, which are important for generating biomass (see ref. 19).

[0010] The present inventors have developed and identified genetic biosensors that are capable of detecting cellular stresses associated with production of recombinant compound of interest, such as proteins or peptides caused for example by inefficient secretion and / or misfolding / aggregation of proteins or peptides in the periplasm or other conditions that causes multiple stress responses in the host cell. It is described herein that the genetic biosensors can be used to screen for conditions that avoid such stress responses, and it is demonstrated that by avoiding stress, increased production titers of secreted and soluble periplasmic compound of interest, such as proteins or peptides can be obtained. It is further demonstrated how dual-colour fluorescence fingerprint obtained from the biosensor nuceotide gene constructs can be used to identify induction conditions that avoid cellular stress and are sustainable to the host cell.

[0011] Accordingly, in a first aspect described herein is a biosensor nucleotide gene construct for monitoring of production of a compond of interest, such as a protein or peptide in a host cell, comprising an inducible promoter responding to a stress factor of the host cell, wherein the promoter is operably linked to a gene encoding a biomarker.

[0012] In a further aspect described herein is a vector comprising the biosensor nucleotide gene construct as described herein.

[0013] In a further aspect described herein is a host cell comprising the biosensor nucleotide gene construct or the expression vector as described herein .

[0014] In a further aspect described herein is a cell culture comprising the host cell described hereinand a growth medium.

[0015] In a further aspect described herein is a method for determining cellular stress in a host cell comprising: a) introducing the biosensor nucleotide gene construct or the vector as described herein into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell so as to allow the host cell to perform a metabolic process producing a compound of interest; c) measuring expression of the biomarker; and d) determining from the level of expression of the biomarker, if the host cell is stressed from its performance of the metabolic process of interest.

[0016] In a further aspect described herein is a method for monitoring production of compound of interest, such as a protein or peptide in a host cell comprising a) introducing the biosensor nucleotide gene construct or the vector as described herein into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell so as to allow it cell to perform a metabolic process producing the compound of interest, such the protein or peptide; c) measuring expression of the biomarker; d) determining at least one parameter of the production of the compound of interest, such the protein or peptide from the level of expression of the biomarker; and e) optionally changing at least one parameter of the metabolic process or the cultivation conditions to change, optionally lower, expression of the biomarker.

[0017] In a further aspect described herein is a method for selecting host cells exhibiting reduced cellular stress comprising: a) introducing the biosensor nucleotide gene construct or the vector as described herein into a host cell performing a metabolic process producing a compound of interest, so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) modifying the host cell by modifying one or more elements of the metabolic pathway of interest to create a library of modified host cells; c) cultivating the library of modified host cell so as to allow it to perform the modified metabolic processes of interest; d) measuring expression of the biomarker in the library of modified host cells upon performing the modified metabolic pathway of interest; ande) selecting modified host cells exhibiting reduced cellular stress by having reduced expression of the biomarker.

[0018] In a final aspect described herein is a biosensor kit of parts for introducing a biosensor into a host cell comprising the biosensor nucleotide gene construct or the vector as described herein and one or more elements selected from a plasmid, competent cells, gene fragment for integration into a vector, primers, enzymes, and / or buffers, and optionally instructions for use.Drawings and figures

[0019] Figure 1 shows a summary of efforts made to amplify the OUTPUT from [PnbpA- fpAsv]. To increase accessibility of the Shine-Dalgarano sequence, the ROSE element (an RNA thermometer) in the 5'UTR of the ibpA promoter was 'unwound' by replacing UCGCU with AAAAA. The polyA variant was not effective in detecting inefficient secretion of MalE, MalEAC and MalE31 when they were induced with increasing concentrations of (w / v) L-arabinose for 3 hours.

[0020] Figure 2 shows that Maltose Binding Protein (MalE / MBP) and misfolding variants can be used as a model system for protein secretion and folding.2A Top, cartoon representation of MalE (the native protein), MalEAC and MalE31 (two misfolding variants) when induced with 0.0002% (w / v) L-arabinose (low expression). All three proteins are efficiently translocated to the periplasm and the signal sequence is removed. MalE folds correctly and is soluble, whereas MalEAC and MalE31 misfold and aggregate. The cleavage of the signal sequence and the solubility were assessed by cell lysis followed by fractionation into soluble and insoluble fractions. The total lysate, soluble and insoluble fractions were analysed by SDS-PAGE and Western blotting with a HisProbe™-HRP Conjugate. The mature form of the protein is denoted M and signal sequence containing version is denoted P. Bottom, cartoon representation of MalE, MalEAC and MalE31 when induced with 0.02% (w / v) L-arabinose (high expression). All three proteins are inefficiently secreted to the periplasm and aggregate.2B Western blots of MalE, MalEAC and MalE31 expressed with increasing concentrations of L- arabinose.2C A per (B) except that samples induced with the same concentration of L-arabinose were separated on the same gel so that the presence or absence of the signal sequence could be determined. The mature form of the protein was identified by comparing mobility in the SDS- PAGE to versions where the signal peptide was not included (AssMalE, AssMalEAC, AssMalEBl). The mature versions are denoted M and the signal sequence containing versions are denoted P.

[0021] Figure 3 shows the identification of biosensor nuceotide gene constructs that can sense stress caused by inefficient secretion and periplasmic misfolding / aggregation.3A Cartoon representation of the [PribpA-gfpAsv] biosensor nuceotide gene construct (26).3B Fluorescence readings from [PnbPA- fpAsv] were taken when preMalE, preMalEAC and preMalE31 were induced for 3 hours with 0% (no induction), 0.0002% (low induction) and 0.02% (w / v) (high induction) of L-arabinose. Fluorescence output was normalised to the ODgoo and then to the 0% sample (i.e. no induction). Ticks and crosses above the graphs indicate whether inefficient secretion, or inclusion body (IB) formation was observed by cell fractionations and Western blotting. Data presented as mean ± standard deviation (s.d.) (n > 3). A statistically significant difference of P < 0.05 is denoted by *, P < 0.01 by **, and P < 0.001 by *** (two-tailed Student's t-test). No statistical difference is denoted n.s.3C Cartoon representations of the [Prcpxp-gfpAsv], [PrCpxp-mcherry sv] and [Prcpxp-mcherryAsv]0PT. biosensor nuceotide gene constructs.3D Fluorescence readings from [Prcpxp-gfpAsv], [PrCpxp-mcherry sv] and [Prcpxp-mcherryAsv]0PTwere taken when preMalE, preMalEAC and preMalE31 were induced for 3 hours with 0% (no induction), 0.0002% (low induction) and 0.02% (w / v) (high induction) L-arabinose. Fluorescence output was normalised to the ODgoo and then to the 0% (no induction) sample. Ticks and crosses above the graphs indicate whether inefficient secretion, or inclusion body (IB) formations was observed by cell fractionations and Western blotting. Data presented as mean ± standard deviation (s.d.) (n > 3). A statistically significant difference of P < 0.05 is denoted by *, P < 0.01 by **, and P < 0.001 by *** (two-tailed Student's t-test). No statistical difference is denoted n.s.

[0022] Figure 4 shows the construction and testing of pQC (plasmid for Quality Control)4A Cartoon representation of the [PnbpA-gfpAsv] / [Prcpxp-mcherryAsv]0PTbiosensor nuceotide gene construct. Transcriptional terminators used to insulate the genetic modules are indicated.4B Fluorescence readings from [PnbpA-gfpAsv] (top panel, white bars) and [Prcpxp-mcherryAsv]0PT(bottom panel, grey bars) were taken when preMalE, preMalEAC and preMalE31 were induced for 3 hours with 0% (no induction), 0.0002% (low induction) and 0.02% (w / v) (high induction) L- arabinose. Fluorescence output was normalised to the ODgoo and then to the 0% (no induction) sample. Ticks and crosses above the graphs indicate whether inefficient secretion, or inclusion body (IB) formation was observed by cell fractionations and Western blotting. Data presented as mean ± standard deviation (s.d.) (n > 3). A statistically significant difference of P < 0.05 is denoted by *, P < 0.01 by **, and P < 0.001 by *** (two-tailed Student's t-test). No statistical difference is denoted n.s.4C Background fluorescence from [PnbPA-gfpAsv] and [Prcpxp-mcherryAsv]0PTwas measured during growth of E. coli. The growth curve is marked in black. Fluorescence measured from [PnbPA-gfpAsv] is marked with a dashed line and that of [Prcpxp-mcherryAsv]0PTis marked with a dotted line. Datapresented as mean ± standard deviation (s.d.) (n > 3).

[0023] Figure 5 shows results where avoiding stress makes bacterial cell factories more efficient.5A Cartoon representation of the plasmids used to express PhoAss-scFvHer2and the [PnbPA- fpAsv] / [Prcpxp-mcherry sv]0PTbiosensor nuceotide gene construct used to monitor stress.5B An overview of the experimental workflow used. PelBss-scFvHer2was expressed with varying concentrations of L-arabinose for 3 hours and the fluorescent 'stress fingerprint' from [PribpA- fpAsv] / [Prcpxp-mcherryAsv]0PTwas captured (white bars and grey bars, respectively). This allowed the identification of inductions condition that were not stressful to the cell.5C A traditional solubility assessment was subsequently carried out on non-stressed and stressed cells that had been induced for 3 hours and 20 hours. Cells were lysed and fractionated. The total lysate, soluble and insoluble fractions were analysed by SDS-PAGE and Western blotting with a HisProbe™-HRP Conjugate.5D Western blots of scFvHer2following fractionation of cells grown with and without stress (left panel). The blots were quantified so that the % soluble protein could be determined (middle panel). [% soluble protein] = [# pixels soluble] / [# pixels soluble] + [# pixels insoluble]. The total soluble yield was also quantified and normalised from the blots (right panel). [Total soluble yield] = [# pixels soluble] / [# pixels soluble_20h No stress]. Data presented as mean ± standard deviation (s.d.) (n > 3). A statistically significant difference of P < 0.05 is denoted by *, P < 0.01 by **, and P < 0.001 by *** (two-tailed Student's t-test). No statistical difference is denoted n.s.

[0024] Figure 6 shows a screen for promoters that can sense stress caused by inefficient secretion and aggregation of MalE, MalEAC and MalE31.6A Cartoon representation of the design used for the biosensor nuceotide gene constructs. The sensors were based on [PribPA-GFP sv] (1) and contained a promoter region upstream of the coding sequence for GFPASV- The nucleotide sequences are available in Supplementary Information, Table S2.6B MalE, MalEAC and MalE31 were induced with 0%, 0.0002%, and 0.02% (w / v) L-arabinose. After 3 hours of expression the fluorescence output from the different biosensor nuceotide gene constructs was recorded and normalised to the ODgoo and then to the 0% sample (i.e. no induction). Promoter and assigned stress response are indicated on the left. Data presented as mean ± standard deviation (s.d.) (n > 3). A statistically significant difference of P < 0.05 is denoted by * (two-tailed Student's t-test). No statistical difference is denoted n.s.6C Background fluorescence from for each sensor was measured during growth of E. coli. The growth curve is marked in black. Fluorescence measured from the sensor is marked with a dashed line. Data presented as mean ± standard deviation (s.d.) (n > 3).

[0025] Figure 7 shows directed evolution of the Translation Initiation Region to generate [Prcpxp- mcherryAsv]0PT.This is a schematic of the workflow used for the directed evolution. PCR with degenerate primers was used to completely randomise 6 nucleotides upstream of the start AUG codon, and synonymously randomised the 2ndand 3rdcodons of the mcherry coding sequence. The library was transformed into cells harbouring pBAD-premalE31 and 190 colonies were picked. preMalE31 was induced with 0.02% (w / v) L-arabinose for 3 hours and fluorescence from the library variants was measured. Fluorescence was normalised to the ODgoo and then to the original [Prcpxp- mcherryAsv] sample. The 'original' [Prcpxp-mcherryAsv] and the 'D4 variant' (renamed [Prcpxp- mcherryAsv]0PT) are marked. The top 15 clones were isolated and re-tested. preMalE31 was induced with either 0% or 0.02% (w / v) L-arabinose respectively and fluorescence output was recorded and normalised to the ODgoo- The top performing clone (D4) was sequenced. The sequences are aligned and differences in nucleotide sequence are marked.

[0026] Figure 8 shows a screen for Cpx-based biosensors that are activated by aggregation periplasmic proteins.Incorporation by reference

[0027] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.DetailsDefinitions

[0028] Abbreviated terms for promoters, cell stress factors, biomarkers, plasmids, primers and test proteins or peptides are those generally known and accepted in the art.

[0029] The term "biomarker" as used herein refers to a reporter molecule capable of generating a signal and reporting an event. In the context of the present decription the biomarker is a compound produced upon inducing a promoter with a cellular factor (compound) whereby the biomarker signals the presence of the cellular factor in the cell.

[0030] The term "stress" or "cell stress" or "cellular stress" as used herein refers to a condition in which a cell experiences disruptions or imbalances in their normal physiological processes, leading to a state of strain or dysfunction. These disruptions can result from various internal or external factors that challenge the cell's ability to maintain its proper functioning and homeostasis. Cellular stress can arise from a variety of sources, including:a) Environmental Factors: Exposure to extreme temperatures, toxins, pollutants, radiation, or chemicals can induce stress in cells; b) Nutritional Imbalances: Inadequate or excessive nutrient availability can stress cells. For instance, nutrient deficiencies or excesses can disrupt cellular metabolism and function; c) Oxidative Stress: This occurs when there's an imbalance between reactive oxygen species (ROS) production and the cell's ability to neutralize them with antioxidants. ROS can damage cellular components such as DNA, proteins, and lipids; d) Genotoxic Stress: This refers to stress caused by DNA damage due to factors like radiation, chemicals, or errors in DNA replication; e) Endoplasmic Reticulum (ER) Stress: The ER is responsible for protein folding and quality control. When misfolded proteins accumulate or when the ER is overwhelmed, it leads to ER stress; f) Heat Shock: Rapid increases in temperature can cause proteins to denature and disrupt cellular processes; g) Hypoxia: Oxygen deprivation can lead to cellular stress, as many cellular processes rely on oxygen for energy production; and h) Mechanical Stress: Physical forces or mechanical distortions can stress cells, particularly those in tissues subjected to mechanical strain.

[0031] Cells have evolved various mechanisms to cope with and respond to stress, collectively known as stress response pathways triggering production of stress response factors. These mechanisms aim to restore cellular homeostasis, repair damage, and promote cell survival. For instance, cells might activate molecular pathways that promote DNA repair, increase antioxidant production, trigger autophagy, or even initiate cell death (apoptosis) if the stress becomes too severe and irreparable.

[0032] Cellular stresses of particular relevance in the field of synthetic biology are stresses caused by accumulation of a compound of interest such as a protein or peptide to be secreted from the cells cytosol, for example by misfolding of proteins in the cell and / or by aggregation of proteins or peptides in the cell.

[0033] The term "stress factor" or "stress response factor" as used herein refers to a compound produced by a cell in response to one or more stresses.

[0019] The term "Green fluorescent Protein" or "GFP" as used herein refers to a protein that exhibits emission of green fluorescence when exposed to ultraviolet (UV) or blue light. This fluorescence is a result of the protein's ability to absorb light energy and then release it as visible green light. GFP and several variants thereof are known in the art.

[0019] The term "mCherry" as used herein refers to a protein that exhibits emission of redfluorescence when exposed to appropriate excitation light. This fluorescence is a result of the protein's ability to absorb light energy and then release it as visible red light. mCherry is known in the art.

[0034] The terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0035] The term "metabolic pathway" as used herein is intended to mean two or more enzymes acting sequentially in a live cell to convert chemical substrate(s) into chemical product(s). Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds such as proteins, for example enzymes (co-enzymes).

[0036] The term "operative biosynthetic metabolic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0037] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0038] The term "and / or" as used herein is intended to represent an inclusive "or". The wording X and / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0039] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences.

[0040] The term "% identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues - x 100 Length of alignment — total number of gaps in alignment

[0041] The term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides- - - x 100Length of alignment — total number of gaps in alignment

[0042] The protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:Cost to open gap: default= 5 for nucleotides / 11 for proteinsCost to extend gap: default = 2 for nucleotides / 1 for proteinsPenalty for nucleotide mismatch: default = -3Reward for nucleotide match: default= 1Expect value: default = 10Wordsize: default = 11 for nucleotides / 28 for megablast / 3 for proteins.

[0043] Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, theidentity calculated in this way is referred to as local identity.

[0044] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0045] The term "vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Vectors can include expression cassettes for the integration of genes into a host cell as well as plasmids, artificial chromosomes and / or chromosomes comprising such genes.

[0046] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present disclosure. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0047] The term "polynucleotide construct" refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0048] The term "operably linked" refers to a configuration in which a control sequence, such as a promoter is placed at an appropriate position relative to the coding polynucleotide (gene) such that the control sequence directs expression of the coding polynucleotide (gene).

[0049] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.

[0050] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0051] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0052] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed disclosure or to imply that certain features are critical, essential, or even important to the structure or function of the itemed disclosure. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0053] The term "cell culture" as used herein refers to a culture medium comprising a plurality ofhost cells of the disclosure. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium ( / .e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.

[0054] It is to be understood that where reference is made throughout this description to sequence identity, a sequence which has X% identity to another sequence, is to be expressly understood as being at least X% identical plus multiple increments of 5% to that other sequence until the identity reaches 100%. For example, where a sequence is at least 70% identical to another sequence, then the sequence is to be understood to also be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 100% identical to that other sequence.

[0055] The term "periplasmic production" as used herein refers to production of a compound of interest, such as a protein or peptide which are synthesized in the cytosol and then secreted to the periplasmic space for adopting their final active form, including but is not limited to: proteins that are synthesized with a signal peptide, proteins that are trafficked through the Sec translocon, proteins that fold in the periplasm with the help of periplasmic chaperones and folding catalysts, and / or proteins that leak out of the periplasmic space and into the media.Biosensor

[0056] The first aspect relates to a biosensor nucleotide gene construct for monitoring of production of a compound of interest such as protein or peptide in a host cell, comprising an inducible promoter responding to a stress factor of the host cell, wherein the promoter is operably linked to a gene encoding a biomarker.

[0057] The compound of interest such as protein or peptide being produced can in principle be any compound, which production can give raise to a stress response in the host cell, though in some embodiments the preferred compound of interest to be monitored is a protein or peptide produced in the periplasm, where the proteins or peptides are finished in the periplasm of the host cell. In this context finishing of a protein or peptide refers to that the protein or peptide may be produced elsewhere in the host cell, while the formation on the final functional protein or peptide occurs in the periplasm. An example of this is proteins or peptide produced as proproteins or propeptides having asignal sequence guiding it to secretion to the cell periplasm, where the signal sequence is cleaved off and the protein or peptide is folded to its functional form optionally with the assistance of one or more chaperone factors.

[0058] Dysfunctional proteins or peptides can cause cellular stress, releasing stress factors and in addition, or alternatively, to any aforementioned embodiments, in some embodiments such stress and stress factors are triggered by dysfunctional / abnormal accumulation, misfolding, aggregation, and / or membrane insertion of the protein or peptide by the host cell. More specifically, in other embodiments the stress factor is triggered by accumulation of compound of interest, such as protein or peptide in the cell cytosol due to dysfunctional compound secretion to the periplasm, by misfolding and / or aggregation of a periplasmic compound, such as protein or peptide in the host cell.

[0059] In particular embodiments the compound of interest, such as protein or peptide, optionally produced or finished in the periplasm of a host cell, is selected from antibodies, antibody fragments, antibody-like fragments, immunogenic proteins suitable for use as immunozing agents, hormones, enzymes, therapeutic proteins and protein fragments to be used a vaccines. More particularly the produced protein can be a hormones such as insulin, human growth hormone (hGH), Erythropoietin (EPO), or Follicle-Stimulating Hormone (FSH). In some embodiments the protein can be a monoclonal Antibody (mAbs) such as Trastuzumab (Herceptin), Rituximab (Rituxan), Adalimumab (Humira), Pembrolizumab (Keytruda), or Infliximab (Remicade). In some embodiments the protein can be a therapeutic enzyme, such as Alteplase (tPA), Pegloticase (Krystexxa), Asparaginase, or Idursulfase (Elaprase). In some embodiments the protein can be a cytokine such as Interferon-alpha, Interleukin- 2 (IL-2), or Granulocyte Colony-Stimulating Factor (G-CSF). In some embodiments the protein can be a blood factor, such as factor VIII, factor IX, or antithrombin III. In some embodiments the protein can be a therapeutic fusion protein, such as Etanercept (Enbrel), or Aflibercept (Eylea). In some embodiments the protein can be a vaccine or vaccine related protein such as recombinant Hepatitis B Surface Antigen, or Human Papillomavirus (HPV) Vaccine Proteins. In some embodiments the peptide can be a therapeutic peptide such as Goserelin (Zoladex), or Exenatide (Byetta).

[0060] In addition, or alternatively, to any aforementioned embodiments, in further embodiments the biosensor construct comprises two or more inducible promoters responding to one or more host cell stress factors, and in more specific embodiments two or more inducible promoters which are different and respond to different host cell stress factors.

[0061] In addition, or alternatively, to any aforementioned embodiments, in further embodiments the inducible promoter of the biosensor construct responds to stress factors triggered by heat shock, dysfunctional secretion of the compound of interest, such as proteins or peptides, to the periplasm, protein misfolding, aggregation, and / or cell envelope stress. In more specific embodiments, theinducible promoter responds cell stress factor triggesed by these stress conditions in particular stress factors selected form G32, GE, Cpx, CpxR, Res, Bae, Psp, and acid stress factor. Accordingly, in preferred embodiments the inducible promoter responds to stress factors comprised in SEQ ID NO: 94 to 100. Inducible promoters that respond to host cell stress factor G32 comprised in SEQ ID NO: 94 and / or CpxR comprised in SEQ ID NO: 96 are particularly useful.

[0062] In addition, or alternatively, to any aforementioned embodiments, in other embodiments the inducible promoter is the PibpA promoter comprised in SEQ ID NO: 3 and / or the Pcpxpromoter comprised in SEQ ID NO: 4 or inducible promoters that respond to host cell stress factors G32 and / or CpxR respectively, having at least 70% identity to said promoter PibPA and / or promoter Pcpxrespectively.

[0063] In addition, or alternatively, to any aforementioned embodiments, in a special embodiment the biosensor construct comprises one or more stress inducible promoters selected from a) PibPA comprised in SEQ ID NO: 3, 78 or 92; b) Pcpxp comprised in SEQ ID NO: 4, 13, 14, 79, 86, 87 or 92; c) Prp0E comprised in SEQ ID NO: 15, 32 or 88; d) PrprA comprised in SEQ ID NO: 16, 33 or 89; e) PpsPA comprised in SEQ ID NO: 17, 34 or 90; f) PhdeA comprised in SEQ ID NO: 18, 35 or 91; g) Pspycomprised in SEQ ID NO: 104; h) Pdegp comprised in SEQ ID NO: 105; i) PyqjA comprised in SEQ ID NO: 106; j) PyqjA. rev comprised in SEQ ID NO: 107; k) PyccA comprised in SEQ ID NO: 112; l) PftnB comprised in SEQ ID NO: 113; m) Pidtc comprised in SEQ ID NO: 114; n) PPsd comprised in SEQ ID NO: 115; o) PsrkA comprised in SEQ ID NO: 116; and / or p) PyebE comprised in SEQ ID NO: 116; or inducible promoters responding to a host cell stress factor comprised in a) SEQ ID NO: 94 (G32); b) SEQ ID NO: 95 (GE); c) SEQ ID NO: 96 (CpxR); d) SEQ ID NO: 97 (RcsB); e) SEQ ID NO: 98 (BaeR);f) SEQ ID NO: 99 (PspF); and / or g) SEQ ID NO: 100 (RcsA), respectively; having at least 50%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as 100% identity to said PibpA, Pcpx, Pcpxp, P rpoE, P rprA, P pspA, PhdeA, P spy, P degP, P yqjA, P yqjA.rev, P yccA- PftnB, Pidtc, Ppsd, PsrkA, or PyebE respectively.

[0064] Particularly biosensor comprising promoters Pspy, Pdegp, PyqjA, or Pyqj.rev, fused to gfpAsv worked well for detecting protein aggregation in the periplasm for for example periplasmic protein MalE31 (see figure 8). Other CpxR-dependent promoters and 5'UTRs were less efficient in biosensors such as PyccA- PftnB, Pidtc, Ppsd, PsrkA, or PyebE- Also P|bpA, Pcpx, Pcpxp proved to be superior promotors in biosensors Over P rpoE, P rprA, P pspA, Or PhdeA-

[0065] In addition, or alternatively, to any aforementioned embodiments, in further embodiments the biosensor construct comprises two or more genes encoding two or more biomarkers operatively linked to two or more inducible promoter(s). The two or more biomarkers may be the same or different and the genes may be operably linked to the the same or different inducible promoters. Where the inducible promoter is the same and the biomarker is the same the biosensor may be more effective in that induction of the promoter triggers expression of more biomarker. Where the inducible promoter is the same and the biomarkers are different the biomarker signal will reflect induction by more than one stress factor. Where the two or more inducible promoters are different and the two or more biomarkers are different, a biomarker signal will be triggered by more than one stress factor and the signal is separable into responses from each stress factor.

[0066] In addition, or alternatively, to any aforementioned embodiments, in a preferred embodiment the two or more biomarker genes encodes different biomarkers and even more preferred the biosensor construct comprises two different inducible promoters responding to two different stress factors of the host cell, wherein the promoters are operably linked to two different genes encoding two different biomarkers.

[0067] In addition, or alternatively, to any aforementioned embodiments, in further embodiments, the biomarker is a fluorescent compound or an enzyme. Suitable fluorescent compounds include Green Fluoresent Protein (GFP), Red Fluoresent Protei, Blue Fluoresent Protein and / or mCherry protein and variants thereof. In some embodiments the Green Fluoresent Protein has a sequence comprised in SEQ ID NO: 5 (GFPASV) and / or the mCherry protein is comprised in SEQ ID NO: 6 (mCherry ASV) or a fluorescent variants thereof having at least 70% identity to GFPASV and / or mCherryAsv respectively.

[0068] In addition, or alternatively, to any aforementioned embodiments, in further embodiments the biosensor construct comprises promoter comprised in SEQ ID NO: 3 (PibpA) and the promotercomprised in SEQ ID NO: 4 (Pcpx ) or inducible promoters responding to host cell stress factors comprised in SEQ ID NO: 94 (o32) and SEQ ID NO: 96 (CpxR) respectively having at least 70% identity to PibpA and Pcpxp respectively, operably linked to genes encoding a first fluorescent biomarker and a second fluorescent biomarker respectively , wherein the first biomarker is different from the second biomarker and wherein optionally the first biomarker is comprised in SEQ ID NO: 5 (GFPASV) and the second biomarker is comprised in SEQ ID NO: 6 (mCherryAsv.), or fluorescent biomarkers having at least 70% identity to said GFPASV and mCherryAsv respectively.

[0069] In addition, or alternatively, to any aforementioned embodiments, in alternative embodiments the biomarker is an enzyme, optionally, selected from R-galactosidase, R-lactamase, and / or luciferase. More particularly enzyme can have a sequence comprised in SEQ ID NO: 101 (R- galactosidase), SEQ ID NO: 102 (R-lactamase), and / or SEQ ID NO: 103 (luciferase) or enzymes having at least 70% identity to said enzymes.

[0070] In addition, or alternatively, to any aforementioned embodiments, in a further embodiment the biosensor construct or promoter comprises one or more Translation Initiation Regions (TIR). The TIR is preferably selected from the TIRs comprised in SEQ ID NO: 108, 109, 110 and / or 111 or a TIR sequence that is at least 70% identical, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% identical to one or more of the TIRs comprised in SEQ ID NO: 108, 108, 110 and / or 111. Particular preferred TIRs are those which comprise a moiety corresponding to residue 6 to 20 (as highlighted below) of SEQ ID NO: 108, 109, 110 and / or 111 or a moiety that is at least 70% identical, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% identical to residue 6 to 20 of SEQ ID NO: 108, 109, 110 and / or Ill. TheTIR's of SEQ ID NO: 109, 110 and 111 were found to work better in biosensors than the TIR of SEQ ID NO: 108.SEQ ID NO: 108 = GGGAG CAAAT GATGA GCAAG GGCGA GSEQ ID NO: 109 = GGGAG ATT AT TATGT CG AAA GGCGA G SEQ ID NO: 110 = GGGAG AACGC TATGT CTAAA GGCGA G SEQ ID NO: 111 = GGGAG GGCTG AATGT CTAAG GGCGA GVectorsThe second aspect relates to a vector comprising the the biosensor nucleotide gene construct as described herein. This vector can be any vector suitable for the host cell and suitable for hosting the biosensor construct and which enables expression of the biomarker. However, specially preferred vectors include chromosomes, artificial chromosomes, and plasmids. Moreover, in someembodiments the vector also comprises a selection cassette or origin of replication. Particularly, the vector is a pSEVA631(Sp) plasmid vector comprising the promoter comprised in SEQ ID NO: 3 (PibpA) and the promoter comprised in SEQ ID NO: 4 (Pcpx) operably linked to genes encoding the biomarker comprised SEQ ID NO: 5 (GFPASV) and the biomarker comprised in SEQ ID NO: 6 (mCherryAsv.) respectively. It is to be understood that where there are two or more inducible promoters operably linked to two or more genes encoding biomarkers the individual construct of an inducible promoter operably linked to a biomarker gene can be located on the same together on the same location or on different locations or on different vectors.Host cells.

[0071] The third aspect relates to a host cell comprising the biosensor nucleotide gene construct or the vector of any preceding embodiments or claims.

[0072] The host cell may be any host cell suitable for and comprising a pathway for production of the compound of interest, such as proteins or peptides and for hosting and expressing the biomarker from the biosensor construct. As the purpose of the biosensor described herein if the detection of cellular stresses caused by production of a compound of interest, in preferred embodiments the host cell is free of genetic modications unrelated to the production of the compound of interest, causing a stress response in itself, i.e. in the absence of production of the compound of interest, such as host cells deprived of genes or expression or genes essential to its normal function and / or survival, such E. coli cells engineered to lack genes encoding phosphatidylserine synthase (pssA), as such a mutations could generate a stress response unrelated to the protein or peptide production for which detection of stress responses is dedired, and thereby may cause a false signals.

[0073] In some embodiments the host cell described herein comprises two biosensor nucleotide gene constructs on the same location on the same vector. In other embodiments the host cell comprises two biosensor nucleotide gene constructs on two different locations on the same vector. In still further embodiments the host cell comprises two biosensor nucleotide gene constructs located on two different vectors.

[0074] In addition, or alternatively, to any aforementioned embodiments, in some embodiments the host cell comprises a combination of two or more stress biosensors inducible by one or more stress factors to either detect more than one stress response or to make detection of a particular stress response with higher precision and / or accuracy.

[0075] The host cell preferably comprises a periplasmic envelope and produced and / or finishes proteins or peptides in this periplasmic envelope, preferably the host cell is genetically engineered to produce a heterologous protein or peptide of interest in the periplasmic envelope. Such proteins or peptides are suitably selected from antibodies, antibody fragments, antibody-like fragments,hormones and / or other recombinant therapeutic proteins or peptides.

[0076] In some embodiments the host cell is a prokaryote cel I, optionally a bacterium, optionally a gram-negative or a gram-positive bacterium. Gram negative bacteria suitable includes those of the genus Escherichia or Pseudomonas, in particular of the species E. coli. Gram positive bacteria suitable includes those of the genus Bacillus, in particular of the species B. subtilis, B. alcolophil is, B. lentus, B. halodurans or B. licheniformis.

[0077] In other embodiments host cell is a eukaryote or archae cell. Preferred eukaryote cells are a fungi or yeasts. Suitable fungi include filamentous fungi, in particular of the genus Aspergillus, such as species A. oryzae, A. niger, or A. aculeatus. Suitable yeasts include those of genus Saccharomyces, Yarrovia or Pichia, such as species P. pastoris, S. carlsbergensis, S. cerevisiae or Y. lipolytica.Culture

[0078] The third aspect relates to a cell culture comprising the host cell described herein and a growth medium. Suitable growth mediums for host cells such as bacterial, fungal and / or yeast cells are known in the art.Methods for using the biosensor construct.

[0079] Further aspects relate to the use of the biosensor construct described herein. Useful applications of the biosensor construct include detection of cellular stresses, monitoring of cells producing proteins or peptides causing cellular stresses and optimizing metabolic pathways to reduce cellular stresses. Accordingly, further provided for herein is a method for determining or detecting cellular stress, preferably stress caused by periplasmic production of a protein or peptide in a host cell comprising a) introducing the biosensor nucleotide gene construct or the vector as described herein into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell so as to allow the host cell to perform a metabolic process of interest; c) measuring expression of the biomarker; and d) determining from the level of expression of the biomarker, if the host cell is stressed from its performance of the metabolic process of interest.

[0080] Applying the biosensor construct to detect if a host cell exhibits stress is very useful for determining if optimixation of metabolic pathways in the host cell is required to reduce cellular stresses.

[0081] Still further provided for herein is a method for selecting host cells exhibiting reduced cellularstress comprising: a) introducing the biosensor nucleotide gene construct or the vector as described herein into a host cell performing a metabolic process of interest producing , so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) modifying the host cell by modifying one or more elements of the metabolic pathway of interest to create a library of modified host cells; c) cultivating the library of modified host cell so as to allow it to perform the modified metabolic processes of interest; d) measuring expression of the biomarker in the library of modified host cells upon performing the modified metabolic pathway of interest; and e) selecting modified host cells exhibiting reduced cellular stress by having reduced expression of the biomarker.

[0062] Applying the biosensor construct to identify modifications in metabolic pathways which reduces cellular stresses is very useful for selecting the optimal production host cell for producing a protein or peptide of interest.

[0082] Still further provided for herein is a method for monitoring production of a protein or peptide of interest in a host cell comprising a) introducing the biosensor nucleotide gene construct or the vector as described herein into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell so as to allow it cell to perform a metabolic process producing the protein or peptide of interest; c) measuring expression of the biomarker; d) determining at least one parameter of the production of the protein or peptide of interest from the level of expression of the biomarker; and e) optionally changing at least one parameter of the metabolic process or the cultivation conditions to change, optionally lower, expression of the biomarker.

[0083] Applying the biosensor construct to perform production control and to monitor cellular stress levels during production is very useful for determing compliance with fermentation conditions and optionally adjusting such fermentation conditions to avoid failure or loss of the fermentation.Kit of parts

[0084] A final aspect relates to a biosensor kit of parts comprising tools materials and / or instruction for using the biosensor construct or the vector in an application of interest, including the detection ofcellular stress, the optimization of metabolic pathways and the monitoring of protein or peptide production, described herein. Accordingly, further provided for herein is a kit of parts comprising the biosensor nucleotide gene construct or the vector described herein, and one or more elements selected from a plasmid, a competent cell, a gene fragment for integration into a vector, a primer, an enzyme, and / or a buffer, and optionally instructions for integrating the biosensor construct into a host cell.Sequence listings

[0085] The present application contains a sequence listing prepared in ST26 compliant format i submitted electronically which is hereby incorporated by reference in its entirety. Further the sequences are described in tables A and B below:Table ATable BExamplesMaterials and methods

[0086] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade.Molecular cloning

[0087] All polymerase change reactions (PCR) were performed using the Q5 High-fidelity DNA polymerase (New England Biolabs). All DNA oligonucleotide primers were synthesised by a commercial provider (Eurofins Genomics (Germany)). Sequences of primers used in these experiments can be found in sequence listing and the sequence section of this description. All DNA sequencing was carried out by commercial provide (Eurofins Genomics (Germany)).Quantification of fluorescence from the biosensor nuceotide gene constructs in bacterial culturesA one millilitre aliquot of a bacterial culture was collected by centrifugation at 3220 x g for 15 min. The culture medium was removed, and the pelleted cells were resuspended in 200 pL of buffer [50mM Tris-HCI pH 8.0, 200mM NaCI and 15mM EDTA], The cell suspension was transferred to a 96-well optical bottom black-wall plate (Thermo Scientific) incubated at room temperature for 3 hours and the fluorescence was determined in a SpectraMax m2e plate reader (Molecular Devices, U.K.). Fluorescence from GFP (biosensor construct [PibpA- fpAsv]) was measured with an excitation wavelength of 475 nm, a filter of 495 nm and emission wavelength of 515 nm. Fluorescence from mCherry (biosensor construct [PCpxp-mcherry sv]) was measured with an excitation wavelength of 585 nm, a filter of 595 nm and emission wavelength of 610 nm. Fluorescence values were normalised by the optical density of the cultures (ODgoo). These values were obtained from a 200 pL aliquot of bacterial culture using a SpectraMax m2e plate reader (Molecular Devices, U.K.) at 600 nm. Finally, the fluorescence per ODgoo was normalised to the control sample (i.e. cells not expressing any recombinant protein).SDS PAGE and Western blottingSDS-PAGE was performed using 1mm 12% tris-glycine acrylamide gels run on the Hoefer Mighty Small II Mini Vertical Electrophoresis System and all gels were run at 100 V for 3 hours (running buffer: 25 mM Tris, 192mM glycine 1% SDS(W / V)). Samples were suspended in Laemmli buffer and boiled at 95°C for 10 minutes prior to loading. The samples consisted of whole cells (1 OD unit / 200 uL Laemmli buffer) or fractionated cells (mixed 3:1 with 4x Laemmli buffer). For Western blotting proteins were transferred to a nitrocellulose membrane using a semidry Trans-Blot SD cell (Bio-Rad) for 30 minutes at 15 V. The membrane was then blocked for 1 to 2 hours or overnight in 5% w / v non-fat milk (PanReac AppliChem) in Tris-buffered saline (TBS) (50 mM Tris, pH 7.4, 200 mM NaCI) before being incubated with the HisProbe-HRP conjugate (15165, Thermo Scientific) at a dilution of 1:10 000 in TBST (TBS supplemented with 1 mL Tween / LTBS) for 1 to 2 hours. The membrane was covered with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and chemiluminecent signal was captured using an Azure c600 Imaging System (Azure Biosystems).Example 1 - Construction of host cells expressing model protein.

[0088] Strains of E. coli strain (MG1655) were modied to produce the model proteins preMalE, preMalE31, preMalEAC, AssMalE, AssMalE31, and AssMalEAC. MalE (Maltose Binding Protein) is a protein which is secreted from the cell cytosol to the periplasm guided by a signal sequence, where the signal sequence is cleaved off and the protein is folded.

[0089] The expression plasmid for producing preMalE was generated by PCR amplification of the known pBAD backbone (ThermoFisher Scientific) using the Pl and P2 primers, and PCR amplification of the malE coding sequence from the E. coli strain MG1655 using the P3 and P4 primers, then assembling them using the in vivo cloning technique as described in ref. 24.

[0090] The expression plasmids for producing preMalE31 were generated by including a 4 base pair mismatch (GAAT to ATCC) in the expression plasmids for producing preMalE using the P5 and P6 primers. This resulted in two amino acid substitutions; G58 to D58 and 159 to P59.

[0091] The construct preMalEAC was generated by PCR using primers P7 and P8. A subsequent cloning mistake was fixed by removing a single base using primers P9 and P10.

[0092] The signal sequences where subsequently removed using primers Pll and P12 to generate plasmids that produced AssMalE, AssMalE31 and AssMalEAC. In all of these expression plasmids the known Tn3.12 cassette, which contained the coding sequence for p-lactamase, was converted to a MINimally expressed Tn3.12MINversion by mismatch PCR using the P13 and P14 primers as described in ref. 25. As a result, these plasmids were maintained using 20 pg / mL ampicillin.Example 2 - Construction of biosensor nucleotide gene constructs and vectors with GFP biomarker and different inducible promoters.

[0093] A pSEVA631(Sp) [PibpA-gfpAsv] biosensor nucleotide gene construct plasmid was constructed using the methods described in ref. 26. A biosensor nucleotide gene construct library (SEQ. ID NO's: 1, 2 and 13 to 21) was created by replacing the promoter region for ibpA (PibpA-) shown in figure 1 with other promoter regions. This was done by PCR amplification of the plasmid backbone using the P15 and P16 primers, and PCR amplification of the promoter sequence from the genome of BL21(DE3) using primers P17-P32, then ligating the fragments using the in vivo cloning technique as described in ref. 24. Promoter regions were chosen as they represented a known stress response (see figure 1) and were defined as the nucleotides annotated as promoters orTF binding sites in RegulonDB as described in ref. 27. These plasmids were maintained using 50 pg / mL spectinomycin.Example 3 - Construction of biosensor nucleotide gene constructs and vectors with mCherry biomarker and inducible promoter.

[0094] A pSEVA631(Sp) [PCpxp-mcherry sv] biosensor nucleotide gene construct plasmid was created by replacing the coding sequence for GFP in example 2 with that of mCherry. This was done by PCR amplification of the plasmid backbone using the P33 and P34 primers, and PCR amplification of the coding sequence for mCherry from pET28-mCherry (28) using primers P35 and P36, then ligating the fragments using the in vivo cloning technique as described in ref. 24.Example 4 - Construction of biosensor nucleotide gene constructs and vectors with both GFP and mCherry biomarkers and inducible promoters.

[0095] A pQC plasmid was created by inserting the [PCpxp-mcherry sv] biosensor nucleotide gene construct into the pSEVA631(Sp) [PibpA- fpAsv] biosensor nuceotide gene construct plasmid of example 2. [Pcpxp-mcherryAsv] was amplified by PCR from pSEVA631(Sp) [PCpxp-mcherry sv] using the P37 and P38 primers. pSEVA631(Sp) [PibpA-gfpAsv] was amplified by PCR using the P39 and P40 primers. The two fragments were ligated (in a back-to back orientation) using the in vivo cloning technique as described in ref. 24. Next the secG terminator was amplified by PCR from the genome of BL21(DE3) using primers P41 and P42. The backbone of pSEVA631(Sp) [PibpA-gfpAsv] [PcPxp-mcherry sv] was amplified by PCR using the P43 and P44 primers. The two fragments were ligated using the in vivo cloning technique as described in ref. 24. Finally, the TIR in [PCpxp-mcherry sv] was changed to the D4 variant by mismatch PCR using primers P45 and P46. The final construct was called pQC.

[0096] The expression plasmids for producing PhoAss-scFvHer2were described previously in ref. 9.Example 5 - Synthetic evolution of the TIR for biosensor nucleotide gene constructs and vectors with mCherry biomarker and inducible promoter.

[0097] Changes in the TIR region of pSEVA631(Sp) [Pcpxp-mcherryAsv] of example 3 were introduced by PCR with degenerate primers. The primers P46 and P47 were used to completely randomise 6 nucleotides upstream of the ATG start codon and synonymously randomised 2 codons downstream of the ATG. The PCR was performed with 30 cycles of 95°C: 30 seconds, 45°C: 30 seconds, and 72°C: 300 seconds. 25 pL of the PCR product was treated with 20 units of Dpnl for 90 minutes before transformation into chemically competent MC1061 cells harbouring pBAD-malE31.

[0098] 190 colonies of the transformants and 2 colonies containing both the original pSEVA631(Sp) [Pcpxp-mcherry sv] and pBAD-malE31 were picked and used to inoculate 500 pL of LB broth containing 50 pg / mL spectinomycin and 20 pg / mL ampicillin in 2.2 mL 96 well plates. These cultures where then grown overnight at 37°C with shaking at 185 rpm. The overnight cultures were back-diluted 1: 100 in 5 mL of LB broth containing antibiotics in 24 well plates. The cultures were grown at 37°C with shaking at 185 rpm to an ODgoo between 0.3 and 0.7. Each culture was then induced with 0.02% (w / v) L- arabinose for 3 hours at 37°C with shaking at 185 rpm. The ODgoo and fluorescence were measured as described below. The plasmids from the top performing 15 clones were purified and used to transform chemically competent MC1061 cells. Three colonies from each transformation (biological replicates) were used to inoculate 500 pL of LB broth containing antibiotics. These cultures where then grown overnight at 37°C with shaking at 185 rpm. Overnight cultures where back diluted 1: 100 to start two sets of 5 mL cultures of LB broth containing antibiotics in 24 well plates. The cultures were grown at 37°C with shaking at 185 rpm to an ODgoo of between 0.3 and 0.7. One set was then induced with 0.02% (w / v) L-arabinose and the other set remained without L-arabinose. These cultures were grown for a further 3 hours at 37°C with shaking at 185 rpm before ODgoo and fluorescence were measured. To isolate the selected pSEVA631(Sp) [Pcpxp-mcherryAsv]0PTplasmid from pBAD-malE31, previously isolated plasmids were transformed into chemically competent MC1061 cells and selected on only 50 pg / mL spectinomycin. Resulting colonies were used to inoculate LB containing just 50 pg / mL spectinomycin or 50 pg / mL spectinomycin and 20 pg / mL ampicillin. Plasmids from cultures that arose from colonies surviving on 50 pg / mL spectinomycin but not 50 pg / mL spectinomycin and 20 pg / mL ampicillin where isolated and sent for sequencing.Example 6 - MalE expression and fractionation of cells

[0099] Coding sequences obtained in examples 1 to 5 cloned into pBAD expression plasmids were transformed into the MC1061 strain (str. K-12 F“ X“ / (ara-leu)7697 [araD139]B / r fcodB- lacl)3 galK16 galE15 el4“ mcrAO relAl rpsL150(StrR) spoTl mcrBl hsdR2(r~m+)), either alone ortogether with a pSEVA631(Sp)-based biosensor nucleotide gene construct plasmid, using a standard heat shock protocol.

[0100] For screening, single colonies were used to inoculate 500 pL LB containing either 20 pg / mL ampicillin (MalE and variants) or 100 pg / mL ampicillin (PhoAss-scFvHer2) in 2.2 mL 96-well plates. When the pSEVA631(Sp)-based biosensor nuceotide gene construct plasmid was present, 50 pg / mL spectinomycin was also added. Cultures were grown at 37°C with shaking at 185 rpm for 16 to 20 hours. A 50 pL aliquot was back-diluted into 5 mL of fresh LB containing appropriate antibiotics in a 5mL 24-well plate. Cultures were grown at 37°C with shaking at 185 rpm to an ODgoo of between 0.3 and 0.7 before induction with varying concentrations of L-arabinose for 3 hours. The ODgoo and fluorescence were measured as described, supra.

[0101] For cell fractionations of MalE and variants, cultures were scaled up to 25 mL in 250 mL Erlenmeyer flasks. Cell pellets were harvested at 3220 x g for 10 min at 4°C. Pellets were re-suspended in 900 pL of Tris-buffer (50 mM Tris, pH 8.3, 100 mM NaCI) and 100 pL of lysozyme from chicken egg white (Sigma Aldrich) followed by a 40-minute incubation at 4°C. Resuspended cells were lysed by sonication using an Ultrasonic processor VCX130 (Sonics & Materials, Inc.) at 10 seconds on / off for 5 min with 70 % intensity. Sonicated samples were centrifuged at 17,000 x g for 3 min at 4°C to remove unbroken cells. A 150 pL aliquot of the supernatant was saved as the total fraction. A 600 pL aliquot of the remaining supernatant was transferred to a Beckman ultracentrifuge tube and centrifuged at 22,000 x g for 1 hour at 4 °C (Optima MAX-XP with TLA55 rotor, Beckman coulter) and a 150 pL aliquot of the supernatant was saved as the soluble fraction. The pellet was resuspended in 600 pL of Trisbuffer, and a 150 pL aliquot was saved as the insoluble fraction.

[0102] For cell fractionations of PhoAss-scFvHer2, cultures were scaled up to 500 mL in IL shaker flasks. Cell pellets were collected by centrifugation at 4000 x g for 20 minutes at 4°C and re-suspended in 50 mL of Tris-buffer (50 mM Tris, pH 8.3, 100 mM NaCI). 50 units of DNAsel were added and the cell suspension was made homogenous using a glass Dounce homogeniser. Cells were lysed by 3 passes through an Avestin emulsiflex C3 high-pressure homogeniser (Avestin, Canada) operating at 10,000 to 15,000 PSI. Un-lysed cells were separated by centrifugation 3220 x g for 10 minutes at 4°C and the pellet was discarded. The total lysate was collected by aspiring a 150 pL aliquot of the lysate. A 35 mL aliquot of the remaining lysate was separated into soluble and insoluble fractions by centrifugation at 22,000 x g for 1 hour at 4°C. An aliquot of the soluble sample was collected by aspiring a 150 pL aliquot of the supernatant. The pellet was re-suspended in 35 mL of Tris-buffer (50 mM Tris, pH 8.3, 100 mM NaCI) and a 150 pL aliquot the insoluble sample was collected.

[0103] To determine the background signal from the biosensor nucleotide gene constructs during growth of the cells, single colonies harbouring pSEVA631-[PibPA-gfpAsv] or pSEVA631-[Pcpxp-gfpAsv] wereinoculated into 5 mL of LB containing 50 pg / mL spectinomycin in 24 well plates. The cultures were grown for 16 to 20 hours at 37°C with shaking at 180 rpm. A 500 pL aliquot was then used to inoculate 50 mL of LB containing 50 pg / mL spectinomycin in 250 mL flasks. The cultures were grown at 37°C with shaking at 180 rpm. At regular time intervals the ODgoo and fluorescence were measured as described, supra.

[0104] preMalE could be expressed in a number of ways, making it an ideal system to create ON and OFF pathway events during the production of periplasmic proteins. At low levels of expression, preMalE was efficiently secreted to the periplasm where the signal peptide is removed, and the mature domain (MalE) folded into a soluble protein (figure 2A, top). At high levels of expression, preMalE was poorly secreted as the Sec translocon becomes saturated. Moreover, it aggregates into cytoplasmic / periplasmic inclusion bodies (figure 2A, bottom).

[0105] preMalE could also be engineered to create OFF pathway events during the production of periplasmic proteins. Two such variants were used; preMalEAC which lacks the C-terminal 94 amino acids and preMalE31 which has Gly-32-Asp and lle-33-Pro mutations. At low levels of expression, both were efficiently secreted to the periplasm where the signal peptide was removed but they subsequently misfolded and aggregated into insoluble inclusion bodies (figure 2A, top). At high levels of expression both were poorly secreted and aggregated into cytoplasmic / periplasmic inclusion bodies (figure 2A, bottom).

[0106] Herein the coding sequences for preMalE, preMalEAC and preMalE31 were cloned into the pBAD24 expression plasmid and expressed by induction with L-arabinose in the MC1061 strain (which cannot metabolise L-arabinose). By increasing the L-arabinose concentration, it was possible to titrate the expression of the proteins (Figure IB). It was also possible to monitor the efficiency of secretion to the periplasm by monitoring the removal of the signal peptide. The latter was done by comparing their mobility in SDS-PAGE to AssMalE, AssMalE31, and AssMalEAC respectively (figure 2C). These data indicate that preMalE, preMalEAC, and preMalE31 did not contain a signal sequence following induction of 0.0002% (w / v) L-arabinose for 3 hours, indicating that they were efficiently secreted to the periplasm. In contrast, a proportion of preMalE, preMalEAC and preMalE31 did contain the signal sequence following induction with 0.002% (w / v) L-arabinose for 3 hours, indicating that they had been partially retained in the cytoplasm.

[0107] To confirm that preMalE, preMalEAC and preMalE31 were folding predictably, cells were fractionated into soluble and insoluble fractions (Figure 2B). Here cells were focussed on that had been induced with 0.0002% (w / v) L-arabinose (low expression) and 0.02% (w / v) L-arabinose (high expression). The data confirmed the solubility profiles presented in the figure cartoons.Example 7 Detecting cellular stress from inefficient secretion from the cytoplasm using [PibpA-gfpAsv]

[0108] It was tested if inefficient secretion of preMalE from the cytoplasm could be monitored. Previous work showed that the heat shock response was activated when proteins with a signal peptide accumulated in the cytoplasm (see ref. 18). Therefore, a biosensor nuceotide gene construct was used that contained a heat shock inducible promoter for the inclusion body binding protein IbpA fused to the coding sequence for an unstable version of the green fluorescent protein [PibpA-gfpAsv] (Figure 3A).

[0109] When preMalE, preMalEAC and preMalE31 were induced with a low concentration of L- arabinose they were efficiently secreted to the periplasm (Figure 2C) and the fluorescence signal from [PibpA-gfpAsv] remained at background levels (Figure 3A). When preMalE, preMalEAC and preMalE31 were expressed with a high concentration of L-arabinose, they accumulated in the cytoplasm (Figure 2C) and the fluorescence from [PibpA-gfpAsv] increased (Figure 3A). These data therefore indicate that [PibpA-gfpAsv] can be used to monitor cellular stress caused by inefficient secretion of proteins from the cytoplasm.

[0110] As the signal from [PibpA-gfpAsv] was 2-fold higher than background, efforts were made to amplify the OUTPUT by translational tuning. Focus was on an RNA thermometer called a ROSE element, which is in the 5'UTR and which represses translation by sequestering the SD sequence. In these experiments attempts were made to make the SD sequence more accessible by varying the sequence of the ROSE element. This approach made [PibpA-gfpAsv] unresponsive to inefficient secretion of preMalE, preMalEAC and preMalE31. The results of these experiments are presented in figure 1.Example 8 - Detecting cellular stress from periplasmic misfolding and aggregation using [Pcpxp- mCherryAsv]

[0111] It was tested if cellular stress caused by misfolding and aggregation of MalE, MalEAC and MalE31 in the periplasm could be monitored. E. coli and other strains possesses several well- characterised cell envelope stress responses (CTE, Cpx, Res, Bae, Psp, acid stress) which detect different physical, chemical and biological stresses in the cell envelope and respond by reprogramming transcription to alleviate the stress. To determine which of these cell envelope stress responses was activated by protein misfolding and aggregation in the periplasm, representative promoters were fused to the coding sequence for an unstable version of the green fluorescent protein as follows; [Prp0E- gfpAsv] (oE), [Pcpxp-gfpAsv] (Cpx), [PrprA-gfpAsv] (Res), [PpspA-gfpAsv] (Psp), and [PhdeA-gfpAsv] (acid stress). Promoters that are activated by multiple stress responses; [Pdegp-gfpAsv] (oE and Cpx), [Pspy-gfpAsv] (Cpx and Bae) were also included (Figure 6; Figure 3C, top).

[0112] When preMalE, preMalEAC and preMalE31 were induced with a high concentration of L- arabinose, they aggregated in the periplasm (Figure 2) and the [Pcpxp-gfpAsv] Biosensor nucleotidegenetic construct was activated (Figure 3D). This observation was consistent with previous work, who showed that expression of preMalE31 elicited a Cpx stress response (see ref. 20). Other biosensor nucleotide gene constructs in the library were not activated (see figure 6). It was also noted that when using [Pcpxp- fpAsv] detection of misfolding and aggregation of preMalE at high expression was low levels even though it was shown to partially misfold and aggregate in the periplasm (figure 2). Further, detection of misfolding and aggregation of preMalE, preMalEAC and preMalE31 at low expression levels was also low. This observation supports a conclusion that it is not misfolding and aggregation being detected, but rather the stress caused by it.

[0113] The [Pcpxp-gfpAsv] biosensor nuceotide gene construct was engineered to (1) make it compatible with downstream applications, and (2) improve its performance. Initially, the coding sequence for GFP was replaced with that of mCherry (Figure 3C, middle), then the OUTPUT signal was amplified by modifying the translation initiation region using a directed evolution approach (Figure 3C, bottom). The directed evolution approach identified a translation initiation region with increased efficiency, by randomising the nucleotides around the AUG start codon for gfpAsv (figure 7). As a consequence, the translational output was increased from the same transcriptional response. The new Biosensor nucleotide gene construct was called [Pcpxp-mcherryAsv]0PT. High-level expression of preMalE, preMalEAC and preMalE31triggered a strong response from [Pcpxp-mcherryAsv]0PT(Figure 3B). Taken together, the data generated indicated that the [Pcpxp-mcherryAsv]0PTis very suitable to detect stress caused by misfolding and aggregation of recombinant proteins in the periplasm.Example 9 - Biosensor nuceotide gene construct for the production of proteins in the periplasm

[0114] A plasmid was engineered that could monitor stress caused by OFF pathway events during the production of periplasmic proteins by cloning the [PibPA-gfpAsv] and [Pcpxp-mcherryAsv]0PTbiosensor nuceotide gene constructs into the pSEVA631(Sp) backbone, separated by the secG leuU terminator (Figure 4A). The plasmid was called pQC (plasmid for Quality Control). Benchmarking of pQC indicated that the resulting fluorescence fingerprint was the same as that obtained when [PibPA-gfpAsv] and [Pcpxp- mcherryAsv]0PTwere tested individually (Figure 4B vs Figure 3).

[0115] The fluorescence from pQC in the absence of recombinant protein production was also monitored at different stages of the growth cycle to better understand the background signal (Figure 4C). [PibPA -gfpAsv] gave a background signal in early exponential phase whereas [PcPxp-mCherry sv]0PTgave a background signal during the stationary phase. At late exponential phase both [PibPA-gfpAsv] and [PcPxp-mCherry sv]0PTgave low background (Figure 4C). These observations indicated that measurements with pQC should ideally be made at late exponential phase.Example 10 - Testing for benefits of avoiding stress during the production of recombinant proteins in the periplasm

[0116] The coding sequence for a single chain antibody fragment that recognises the human epidermal growth factor (scFvHer2) was cloned downstream of the coding sequence of a PhoA signal peptide, in the pBAD24 expression plasmid. PhoAss-scFvHer2was then expressed in MC1061 cells containing pQC (Figure 5A). Induction conditions that avoided stress were identified by inducing the cultures for 3 hours with increasing concentrations of L-arabinose and monitoring the fluorescence fingerprint from pQC (Figure 5B). At 0.0002% (w / v) L-arabinose (and lower concentrations) the fluorescence levels were comparable with those obtained from uninduced cells, indicating that the cells were not stressed by the production of scFvHer2(Figure 5B). At 0.002% (w / v) L-arabinose (or higher concentrations) the fluorescence levels from pQC were higher than those obtained from uninduced cells, indicating that these cells were stressed by the production of scFvHer2.

[0117] To evaluate the effects of stress, cells were induced with 0.0002% and 0.2% (w / v) L-arabinose (non-stressed and stressed, respectively) and harvested after 3 or 20 hours of induction. The yields of secreted and soluble scFvHer2were estimated using a traditional solubility assessment. This involved cell lysis then fractionation, SDS-PAGE and Western blotting (Figure 5C). In non-stressed cells, approximately 40% of the scFvHer2produced was soluble after a 3-hour induction, indicating that scFvHer2was prone to aggregation. However, after 20-hours of induction approximately 80% of the scFvHer2produced was soluble (Figure 5D). This observation indicated that, in the absence of stress, the cells are able to accumulate soluble scFvHer2. In stressed cells, approximately 30% of the scFvHer2produced was soluble after a 3-hour induction. However, after 20-hours of induction approximately 10% of the scFvHer2produced was soluble (Figure 4D). This observation indicated that, in the presence of stress, the cells accumulated insoluble protein scFvHer2. Notably the total amount of secreted and soluble scFvHer2obtained was highest in non-stressed cells after 20 hours of induction (Figure 5D).Further conclusions

[0118] The production of periplasmic proteins in high titres is a challenging endeavour for those working in both academia and industry. It hinders the advancement of fundamental knowledge, as well as the commercialisation of biotech-based solutions and new therapeutic targets (hormones, antibody fragments etc). Previous work has identified two major OFF-pathway events in the production of a recombinant periplasmic protein that need to be avoided: (1) inefficient secretion from the cytoplasm and (2) misfolding and aggregation in the periplasm. These OFF-pathway events cause cellular stress, which initiates a program of transcriptional re-wiring that helps the cell regain homeostasis. Stress induced transcriptional reprogramming is detrimental to recombinant proteinproduction as it affects the ability of the cell to sustain protein synthesis. It also affects cellular energy metabolism and cell growth, which are important for generating biomass.

[0119] A simple strategy to avoid production stress is to tune the expression levels of the recombinant protein to the capacity of the cell using inducible promoters or genetic circuits. It is also possible to manipulate the culture conditions (time, temperature, media) or use protein engineering strategies such as solubility tags or co-expression of chaperones. To implement these approaches effectively, it is necessary to identify relevant stresses, monitor them, and avoid activating them.

[0120] The present inventors have shown that an engineered a biosensor nuceotide gene construct plasmid such as pQC (plasmid for Quality Control) can monitor two common stresses that occur during the production of recombinant proteins in the periplasm. pQC was constructed from: (1) the [PjbpA- gfpAsv] biosensor nucleotide gene construct, which monitors the heat shock response (o32) and is activated when proteins are not efficiently secreted from the cytoplasm, and (2) the [Pcpxp- mcherryAsv]0PTgenetic module (described here), which monitors the Cpx extra-cytoplasmic stress response and is activated when proteins misfold / aggregate in the periplasm. pQC was evaluated by titrating the expression levels of preMalE, preMalE'c, preMalE31and PhoAss-scFvHer2, then monitoring stress using a simple dual-colour fluorescent fingerprint. Using this approach, we were able to identify induction conditions that did not stress the cell, as well as those that did. With all recombinant proteins tested we observed that activation of both [PibPA-gfpAsv] and [PcPxp-mcherry sv]0PToccurred at approximately the same induction conditions. Thus, it was not possible to tease apart stress caused by inefficient secretion from the cytoplasm, from stress caused by misfolding and aggregation in the periplasm. The current data therefore indicate that, above a certain induction condition, both the secretion and folding capacity of the cell was exceeded. This capacity could be increased using protein engineering strategies (i.e. signal peptides, solubility tags, co-expression of chaperones) (8,14,15). Although we have not explored these possibilities here, we believe that pQC would be useful as a screening tool when evaluating these strategies.

[0121] A key finding of these experiments was that avoiding stress resulted in higher production titres of soluble and secreted scFvHer2. In these experiments we used pQC to identify induction conditions that did not stress the cell, as well as those that did, then compared the quantity of secreted and soluble protein obtained. Over a 20-hour induction we obtained >10-times more soluble and secreted scFvHer2using the non-stressed conditions. The data are consistent with other published studies, which have used biochemical approaches to demonstrate that harmonising the production rate with the capacity of the Sec translocon results in higher production titres of recombinant proteins.

[0122] One aspect of these experiments is the approach used to amplify the OUTPUT signal from [PcPxp-mcherryAsv]. Promoter regions derived from nature (like PjbPA- and Pcpxp-) have notoriously subtletranscriptional responses, as this is what is required in the context of the cell. For bioengineering and industrial applications, a stronger more robust OUTPUT is usually desired. In these experiments we used a directed evolution approach to optimise the efficiency of the TIR in [Pcpxp-mcherryAsv]. The approach aimed to increase the efficiency of translation without affecting the transcriptional response. We observed that the OUTPUT signal from [Pcpxp-mcherryAsv]0PTwas 2 to 3-fold higher than that from [Pcpxp-mcherry sv]. This methodology has been developed by the present inventors and has been used previously to amplify the OUTPUT signal from the T7 and pBAD promoters. It is anticipated that it will be useful for other transcription-based biosensors.

[0123] Finally, it should be noted that the biosensor nucleotide gene constructs incorporated in eg, pQC can also be used for monitoring the misfolding and aggregation of cytoplasmic and membrane proteins, as well as the metabolic burden associated with recombinant protein production.Example 11 - Screening for further promoters responding to stress caused by periplasmic protein production

[0124] Four further promoters Pspy, PdegP, PyqjA or PyqjA.rev (SEQ ID NO: 104-107) were identified as CpxR-dependent promoter and fused to gfpAsv (SEQ ID NO: 5) in a biosensor including 5'UTR these biosensors were highly useful in detecting stress caused by aggregation of a model protein, MalE31, in the periplasm, using similar experimental setup as described in the previous examples, in particular when also including the TIR's of SEQ ID NO: 109, 110 or 111 providing enhanced response to stress factors. In the same experiment promoters PyccA, PftnB, PldtC, Ppsd, PsrkA and PyebE were less effective. MalE31 was induced with 0% and 0.02% (w / v) L-arabinose (white and dark bars, respectively). After 3 hours of expression the fluorescence output from the different biosensors was recorded and normalised to the ODgoo and then compared to the 0% sample (i.e. no induction). Promoter and 5'UTR used in the biosensor is indicated in figure 8. Data are presented as mean ± standard deviation (s.d.) (n> 3). A statistically significant difference of P < 0.05 is denoted by * , P < 0.01 is denoted by ** (two-tailed Student's t-test). No statistical difference is denoted n.s. Note that PyqjA gave a positive response when MalE31 was expressed with higher arabinose concentrations.References1. Rosano GL, Morales ES, Ceccarelli EA. New tools for recombinant protein production in Escherichia coli : A 5-year update. Protein Science. 2019 Aug;28(8):1412-22.2. Rong Y, Jensen SI, Lindorff-Larsen K, Nielsen AT. Folding of heterologous proteins in bacterial cell factories: Cellular mechanisms and engineering strategies. Biotechnology Advances. 2023 Mar;63:108079.3. Mergulhao FJM, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnology Advances. 2005 May;23(3):177-202.4. Karyolaimos A, de Gier JW. Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli. Front Bioeng Biotechnol. 2021 Dec 14;9:797334.5. Arbabi-Ghahroudi M, Tanha J, MacKenzie R. Prokaryotic expression of antibodies. Cancer Metastasis Rev. 2005 Dec;24(4):501-19.6. Kadokura H, Beckwith J. Mechanisms of Oxidative Protein Folding in the Bacterial Cell Envelope. Antioxidants & Redox Signaling. 2010 Oct 15;13(8):1231-46.7. Denoncin K, Collet JF. Disulfide Bond Formation in the Bacterial Periplasm: Major Achievements and Challenges Ahead. Antioxidants & Redox Signaling. 2013 Jul;19(l):63-71.8. Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 2004 Nov;22(ll):1399-408.9. Mirzadeh K, Shilling PJ, Elfageih R, Cumming AJ, Cui HL, Rennig M, et al. Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microb Cell Fact. 2020 Apr 7;19(1):85.10. Park S, Liu G, Topping TB, Cover WH, Randall LL. Modulation of Folding Pathways of Exported Proteins by the Leader Sequence. Science. 1988 Feb 26;239(4843):1033-5.11. Beena K, Udgaonkar J B, Varadarajan R. Effect of Signal Peptide on the Stability and Folding Kinetics of Maltose Binding Protein. Biochemistry. 2004 Mar l;43(12):3608-19.12. Smets D, Tsirigotaki A, Smit JH, Krishnamurthy S, Portaliou AG, Vorobieva A, et al. Evolutionary adaptation of the protein folding pathway for secretability. The EMBO Journal [Internet], 2022 Dec [cited 2022 Dec 30];41(23). Available from: https: / / onlinelibrary.wiley.com / doi / 10.15252 / embj.2022111344.13. Tsirigotaki A, De Geyter J, Sostaric' N, Economou A, Karamanou S. Protein export through the bacterial Sec pathway. Nat Rev Microbiol. 2017 Jan;15(l):21-36.14. Stull F, Betton JM, Bardwell JCA. Periplasmic Chaperones and Prolyl Isomerases. Slauch JM, Ehrmann M, editors. EcoSal Plus. 2018 Feb 8;8(l):ecosalplus. ESP-0005-2018.15. De Geyter J, Tsirigotaki A, Orfanoudaki G, Zorzini V, Economou A, Karamanou S. Protein folding in the cell envelope of Escherichia coli. Nat Microbiol. 2016 Jul 26;l(8):16107.16. Horga LG, Hal liwel I S, Castineiras TS, Wyre C, Matos CFRO, Yovcheva DS, et al. Tuning recombinant protein expression to match secretion capacity. Microb Cell Fact. 2018 Dec;17(l):199.17. Schlegel S, Rujas E, Ytterberg AJ, Zubarev RA, Luirink J, de Gier JW. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels. Microb Cell Fact. 2013 Mar 12;12:24.18. Wild J, Walter WA, Gross CA, Altman E. Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response. J Bacteriol. 1993 Jul;175(13):3992-7.19. Baumgarten T, Ytterberg AJ, Zubarev RA, de Gier JW. Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress. Atomi H, editor. Appl Environ Microbiol. 2018 Jun 15;84(12):e00270-18.20. Hunke S, Betton JM. Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli: Temperature effect on inclusion body formation. Molecular Microbiology. 2003 Nov ll;50(5): 1579-89.21. Karyolaimos A, Ampah-Korsah H, Hillenaar T, Mestre Borras A, Dolata KM, Sievers S, et al. Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening. Front Microbiol. 2019;10:1511.22. Structural Genomics Consortium, Architecture et Fonction des Macromolecules Biologiques, Berkeley Structural Genomics Center, China Structural Genomics Consortium, Integrated Center for Structure and Function Innovation, Israel Structural Proteomics Center, et al. Protein production and purification. Nat Methods. 2008 Feb;5(2):135-46.23. Cornvik T, Dahlroth SL, Magnusdottir A, Herman MD, Knaust R, Ekberg M, et al. Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli. Nat Methods. 2005 Jul;2(7):507-9.24. Watson JF, Garci'a-Nafn'a J. In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning. Journal of Biological Chemistry. 2019 Oct;294(42):15271-81.25. Cumming AJ, Khananisho D, Harris R, Bayer CN, Nprholm MHH, Jamshidi S, et al. Antibiotic-Efficient Genetic Cassette for the TEM-1 -Lactamase That Improves Plasmid Performance. ACS Synth Biol. 2022 Jan 21;ll(l):241-53.26. Zutz A, Hamborg L, Pedersen LE, Kassem MM, Papaleo E, Koza A, et al. A dual-reporter system for investigating and optimizing protein translation and folding in E. coli. Nat Commun. 2021 Oct 19;12(l):6093.Santos-Zavaleta A, Salgado H, Gama-Castro S, Sanchez-Perez M, Gomez-Romero L, Ledezma-Tejeida D, et al. RegulonDB v 10.5: tackling challenges to unify classic and high throughput knowledge of gene regulation in E. coli K-12. Nucleic Acids Research. 2019 Jan 8;47(Dl):D212-20.28. Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Kock Z, Daley DO. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol. 2020 May 7;3(1):214.29. Chen J. Molecular mechanism of the Escherichia coli maltose transporter. Current Opinion in Structural Biology. 2013 Aug;23(4):492-8.30. Bassford PJ. Export of the periplasmic maltose-binding protein ofEscherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):401-39.31. Walters BT, Mayne L, Hinshaw JR, Sosnick TR, Englander SW. Folding of a large protein at high structural resolution. Proc Natl Acad Sci USA. 2013 Nov 19;110(47):18898-903.32. Duplay P, Szmelcman S, Bedouelle H, Hofnung M. Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. Journal of Molecular Biology. 1987 Apr;194(4):663- 73.33. Betton JM, Sassoon N, Hofnung M, Laurent M. Degradation versus Aggregation of Misfolded Maltose-binding Protein in the Periplasm of Escherichia coli. Journal of Biological Chemistry. 1998 Apr;273(15):8897-902.34. Betton JM, Hofnung M. Folding of a Mutant Maltose-binding Protein of Escherichia coli Which Forms Inclusion Bodies. Journal of Biological Chemistry. 1996 Apr;271(14):8046-52.35. Saul FA. Crystal structure of a defective folding protein. Protein Science. 2003 Mar 1;12(3):577- 85.36. Spurlino JC, Lu GY, Quiocho FA. The 2.3-A resolution structure of the maltose- or maltodextrin- binding protein, a primary receptor of bacterial active transport and chemotaxis. J Biol Chem. 1991 Mar 15;266(8):5202-19.37. Duplay P, Szmelcman S, Bedouelle H, Hofnung M. Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. I. Transport of maltose. J Mol Biol. 1987 Apr 20;194(4):663-73.38. Betton JM, Hofnung M. Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. J Biol Chem. 1996 Apr 5;271(14):8046-52.39. Bury-Mone S, Nomane Y, Reymond N, Barbet R, Jacquet E, Imbeaud S, et al. Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli. Matic I, editor. PLoS Genet. 2009 Sep 18;5(9):el000651.40. Mirzadeh K, Martinez V, Toddo S, Guntur S, Herrgard MJ, Elofsson A, et al. Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction. ACS Synth Biol. 2015 Sep 18;4(9):959-65.41. Chen YJ, Liu P, Nielsen AAK, Brophy JAN, Clancy K, Peterson T, et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods. 2013 Jul;10(7):659-64.42. De Wulf P, Kwon O, Lin ECC. The CpxRA Signal Transduction System of Escherichia coli : Growth- Related Autoactivation and Control of Unanticipated Target Operons. J Bacteriol. 1999 Nov;181(21):6772-8.43. Karyolaimos A, Ampah-Korsah, Hilenaar, Tamara, Mestre Borras, Anna, Dolata, Katarzyna Magdalena, Sievers, Susanne, et al. Enhancing recombinant protein yields in the E. coli periplasm by combining signal peptide and production rate screening. Frontiers in Microbiology. 2019;10(1511).44. Horga LG, Hal liwel I S, Castineiras TS, Wyre C, Matos CFRO, Yovcheva DS, et al. Tuning recombinant protein expression to match secretion capacity. Microb Cell Fact. 2018 Dec;17(l):199.45. Ceroni F, Boo A, Furini S, Gorochowski TE, Borkowski O, Ladak YN, et al. Burden-driven feedback control of gene expression. Nat Methods. 2018 May;15(5):387-93.46. Daley D, Mirzadeh K, Toddo S, Guntur S. Selective optimisation of a ribosome binding site for protein production [Internet], EP3234146; US10,696,963, 2016 [cited 2018 Mar 29], Available from: https: / / patents.google.com / patent / WO2016099388Al / en47. Shilling PJ, Khananisho D, Cumming AJ, Sbderstrbm B, Daley DO. Signal amplification of araC pBAD using a standardized translation initiation region. Synthetic Biology. 2022 Jul 26;7(l):ysac009.48. Shimohata N, Chiba S, Saikawa N, Ito K, Akiyama Y. The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site: Membrane protein stress and proteases. Genes to Cells. 2002 Jul;7(7):653-62.49. Guidi C, De Wannemaeker L, De Baets J, Demeester W, Maertens J, De Paepe B, et al. Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli. Microb Cell Fact. 2022 Dec 15;21(l):260.50. Zutz A, Hamborg L, Pedersen LE, Kassem MM, Papaleo E, Koza A, et al. A dual-reporter system for investigating and optimizing protein translation and folding in E. coli. Nat Commun. 2021 Oct 19;12(l):6093.51. Bury-Mone S, Nomane Y, Reymond N, Barbet R, Jacquet E, Imbeaud S, et al. Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli. Matic I, editor. PLoS Genet. 2009 Sep 18;5(9):el000651.Items of the disclosureThe present disclosure further provides the following embodiments and items:1. A biosensor nucleotide gene construct for monitoring of periplasmic protein or peptide production in a host cell, comprising an inducible promoter responding to a stress factor of the host cell, wherein the promoter is operably linked to a gene encoding a biomarker.2. The construct of item 1 comprising the PibpA promoter comprised in SEQ ID NO: 3 and / or the Pcpxpromoter comprised in SEQ ID NO: 4 or inducible promoters having at least 70% identity to promoter PibPA and / or promoter Pcpxresponding to a host cell stress factor comprised in SEQ ID NO: 94 (s32) orSEQ ID NO: 96 (CpxR) respectively.3. The construct of any preceding item wherein the biomarker is a Green Fluoresent Protein (GFP) and / or mCherry protein, wherein optionally the Green Fluoresent Protein has a sequence comprised in SEQ ID NO: 5 (GFPASV) and / or the mCherry protein is comprised in SEQ ID NO: 6 (mCherry sv) or a fluorescent biomarker having at least 70% identity to GFPASV and / or mCherryAsv respectively.4. A vector comprising the biosensor nucleotide gene construct of any preceding item.5. The vector of item 4 wherein the vector is a chromosome, an artificial chromosome, or a plasmid.6. A host cell comprising the biosensor nucleotide gene construct or the vector of any preceding item.7. The host cell of item 6 comprising two biosensor nucleotide gene constructs located on two different locations on the same vector.8. The host cell of item 6 comprising two biosensor nucleotide gene constructs at two different vectors.9. The host cell of any of items 6 to 8 wherein the host cell is an Escherichia coli cell genetically engineered to produce a heterologousprotein or peptide of interest in the periplasmic envelope.10. The host cell of items 9 wherein the heterologous protein or peptide of interest is selected from antibodies, antibody fragments, antibody-like fragments, hormones or recombinant therapeutic proteins or peptides.11. A cell culture comprising the host cell of any of items 6 to 10 and a growth medium.12. A method for determining cellular stress in a host cell comprising: a) introducing the biosensor nucleotide gene construct of any of items 1 to 3 or the vector of items 4 to 5 into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell so as to allow the host cell to perform a metabolic process of interest; c) measuring expression of the biomarker; andd) determining from the level of expression of the biomarker if the host cell is stressed from its performance of the metabolic process of interest.13. A method for monitoring production of a protein or peptide of interest in a host cell comprising a) introducing the biosensor nucleotide gene construct of any of items 1 to 3 or the vector of items 4 to 5 into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) cultivating the host cell to allow it cell to perform a metabolic process producing the protein or peptide of interest; c) measuring expression of the biomarker; d) determining at least one parameter of the production of the protein or peptide of interest from the level of expression of the biomarker; and e) optionally changing at least one parameter of the metabolic process or the cultivation conditions to change, optionally lower, expression of the biomarker.14. A method for selecting host cells exhibiting reduced cellular stress comprising: a) introducing the biosensor nucleotide gene construct of any of items 1 to 3 or the vector of items 4 to 5 into a host cell performing a metabolic process of interest, so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) modifying the host cell by modifying one or more elements of the metabolic pathway of interest to create a library of modified host cells; c) cultivating the library of modified host cell to allow it to perform the modified metabolic processes of interest; d) measuring expression of the biomarker in the library of modified host cells upon performing the modified metabolic pathway of interest; and e) selecting modified host cells exhibiting reduced cellular stress by having reduced expression of the biomarker.15. A biosensor kit of parts for introducing a biosensor into a host cell comprising the biosensor nucleotide gene construct of items 1 to 3 or the vector of any of items 4 to 5 and one or more elements selected from a plasmid, competent cells, gene fragment for integration into a vector, primers, enzymes, and / or buffers, and optionally instructions for use.* * *

Claims

Claims1. A biosensor nucleotide gene construct for monitoring of production of a compound of interest in a host cell, comprising an inducible promoter responding to a stress factor of the host cell, wherein the promoter is operably linked to a gene encoding a biomarker.

2. The construct of claim 1 wherein the compound of interest is a protein or peptide.

3. The construct of claim 1 wherein the compound of interest is selected from antibodies, antibody fragments, antibody-like fragments, immunogenic proteins suitable for use as immunozing agents, hormones, enzymes, therapeutic proteins or peptides.

4. The construct of any preceding claim wherein the production is periplasmic production.

5. The construct of any preceding claim wherein the host cell stress factor is triggered by accumulation, misfolding, aggregation, and / or membrane insertion of the compound of interest by the host cell.

6. The construct of any preceding claim wherein the host cell stress factor is triggered by cytosolic accumulation of a compound of interest secreted to the periplasm, by misfolding and / or aggregation of a periplasmic compound of interest in the host cell.

7. The construct of any preceding claim comprising two or more inducible promoters responding to one or more host cell stress factors.

8. The construct of claim 7 wherein the two or more inducible promoters are different and respond to different host cell stress factors.

9. The construct of anyone of claim 7 to 8 wherein the inducible promoter responds to stress factors triggered by heat shock, dysfunctional secretion from the cytosol, dysfunctional compound of interest misfolding, aggregation, and / or cell envelope stress.

10. The construct of anyone of claim 7 to 9 wherein the inducible promoter responds to a host cell stress factor selected from s32, sE, Cpx, CpxR, Res, Bae, Psp, and acid stress factor.

11. The construct of anyone of claim 7 to 10 wherein the inducible promoter responds to a host cellstress factor comprised in anyone of SEQ ID NO: 94 to 100.

12. The construct of anyone of claim 11 wherein the inducible promoter responds to host cell stress factor s32 comprised in SEQ ID NO: 94 and / or CpxR comprised in SEQ ID NO: 96.

13. The construct of any preceding claim comprising the PibpA promoter comprised in SEQ ID NO: 3 and / or the Pcpx promoter comprised in SEQ ID NO: 4 or inducible promoters responding to a host cell stress factor having at least 70% identity to promoter PibPA and / or promoter Pcpx.

14. The construct of any preceding claim comprising inducible promoters selected from e) PibPA comprised in SEQ ID NO: 3, 78 or 92; f) PcpxP comprised in SEQ ID NO: 4, 13, 14, 79, 86, 87 or 92; g) PrpoE comprised in SEQ ID NO: 15, 32 or 88; h) PrprA comprised in SEQ ID NO: 16, 33 or 89; i) PpspA comprised in SEQ ID NO: 17, 34 or 90; j) PhdeA comprised in SEQ ID NO: 18, 35 or 91; k) Pspy comprised in SEQ ID NO: 104; l) PdegP comprised in SEQ ID NO: 105; m) PyqjA comprised in SEQ ID NO: 106; n) PyqjA.rev comprised in SEQ ID NO: 107; o) PyccA comprised in SEQ ID NO: 112; p) PftnB comprised in SEQ ID NO: 113; q) PldtC comprised in SEQ ID NO: 114; r) Ppsd comprised in SEQ ID NO: 115; s) PsrkA comprised in SEQ ID NO: 116; and / or t) PyebE comprised in SEQ ID NO: 116; or inducible promoters responding to a host cell stress factor comprised in a)SEQ ID NO: 94 (s32); b)SEQ ID NO: 95 (sE); c)SEQ ID NO: 96 (CpxR); d)SEQ ID NO: 97 (RcsB); e)SEQ ID NO: 98 (BaeR); f)SEQ ID NO: 99 (PspF); and / or g)SEQ ID NO: 100 (RcsA) respectively.having at least 50%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as 100% identity to said PibpA, Pcpx, Pcpxp, PrpoE, P rprA, P pspA, PhdeA , P spy, P degP, P yqjA, P yqjA.rev, P yccA- PftnB, Pidtc, Ppsd, PsrkA, or PyebE respectively.

15. The construct of any preceding claim further co prising one or more T ranslation Initiation Regions (TIR) comprising a moiety corresponding to residue 6 to 20 of SEQ ID NO: 108, 109, 110 and / or 111 or a moiety that is at least 70% identical, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% identical to residue 6 to 20 of SEQ ID NO: 108, 109, 110 and / or 111.

16. The construct of claim 15 further comprising one or more Translation Initiation Regions (TIR) selected from the TIRs comprised in SEQ ID NO: 108, 109, 110 and / or 111 or a TIR sequence that is at least 70% identical, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% identical to one or more of the TIRs comprised in SEQ ID NO: 108, 108, 110 and / or 111.

17. The construct of any preceding claim comprising two or more genes encoding two or more biomarkers operatively linked to the inducible promoter.

18. The construct of claim 17 wherein the two or more biomarker genes encodes different biomarkers.

19. The construct of anyone of claim 17 to 18 comprising two different inducible promoters responding to two different stress factors of the host cell, wherein the promoters are operably linked to two different genes encoding two different biomarkers.

20. The construct of anyone of claim 17 to 19 wherein the biomarker is a fluorescent compound or an enzyme.

21. The construct of claim 20 wherein fluorescent compound is Green Fluoresent Protein (GFP) and / or mCherry protein.

22. The construct of claim 21 wherein the Green Fluoresent Protein has a sequence comprised in SEQ ID NO: 5 (GFPASV) and / or the mCherry protein is comprised in SEQ ID NO: 6 (mCherryAsv) or a fluorescent biomarker having at least 70% identity to GFPASV and / or mCherryAsv respectively.

23. The construct of any preceding claim comprising the promoter comprised in SEQ ID NO: 3 (PibpA) and the promoter comprised in SEQ ID NO: 4 (Pcpx ) or inducible promoters responding to host cell stress factors comprised in SEQ ID NO: 94 (s32) and SEQ ID NO: 96 (CpxR) respectively having at least 70% identity to PibPA and Pcpxp respectively, operably linked to genes encoding a first fluorescent biomarker and a second fluorescent biomarker respectively , wherein the first biomarker is different from the second biomarker and wherein optionally the first biomarker is comprised in SEQ ID NO: 5 (GFPASV) and the second biomarker is comprised in SEQ ID NO: 6 (mCherryAsv.), or fluorescent biomarkers having at least 70% identity to said GFPASV and mCherryAsv respectively.

24. The construct of anyone of claim 17 to 19 wherein the enzyme is selected from R-galactosidase, R-lactamase, and / or luciferase.

25. The construct of any proceeding claim wherein the enzyme has a sequence comprised in SEQ ID NO: 101 (R-galactosidase), SEQ ID NO: 102 (R-lactamase), and / or SEQ ID NO: 103 (luciferase) or an enzyme having at least 70% identity to said enzymes.

26. A vector comprising the the nucleotide gene construct of any preceding claim.

27. The vector of claim 26 wherein the vector is a chromosome, an artificial chromosome, or a plasmid.

28. The vector of anyone of claim 26 to 27 further comprising a selection cassette or origin of replication.

29. The vector of anyone of claim 26 to 28 wherein the vector is a pSEVA631(Sp) plasmid vector comprising the promoter comprised in SEQ ID NO: 3 (PibPA) and the promoter comprised in SEQ ID NO: 4 ( PcPx) operably linked to genes encoding the biomarker comprised SEQ ID NO: 5 (GFPASV ) and the biomarker comprised in SEQ ID NO: 6 (mCherryAsv.) respectively.

30. A host cell comprising the biosensor nucleotide gene construct or the vector of any preceding claim.

31. The host cell of claim 30 comprising two biosensor nucleotide gene constructs located on two different locations on the same vector.

32. The host cell of claim 30 comprising two biosensor nucleotide gene constructs at two different vectors.

33. The host cell of anyone of claims 30 to 32 comprising a periplasmic envelope.

34. The host cell of claims 33 wherein the host cell is genetically engineered to produce a heterologous compound of interest, optionally a protein or peptide, optionally in the periplasmic envelope.

35. The host cell of anyone of claim 34 wherein the heterologous compound of interest is selected from antibodies, antibody fragments, antibody-like fragments, immunogenic proteins suitable for use as immunozing agents, hormones, enzymes or recombinant therapeutic proteins or peptides.

36. The host cell of anyone of claims 30 to 35 wherein the host cell is a prokaryote cell.

37. The host cell of claims 36 wherein the prokaryote cell is a bacterial cell.

38. The host cell of claims 37 wherein the bacterial cell is a gram-negative bacterial cell.

39. The host cell of claims 38 wherein the gram-negative bacterial cell is of the genus Escherichia or Pseudomonas.

40. The host cell of claims 39 wherein the Escherichia bacterium is of the species E. coli.

41. The host cell of claims 37 wherein the bacterial cell is a gram-positive bacterial cell.

42. The host cell of claims 41 wherein the gram-positive bacterial cell is of the genus Bacillus.

43. The host cell of claims 42 wherein the Bacillus bacterium is of the species subtilis alcolophilis, lentus, halodurans or licheniformis.

44. The host cell of anyone of claims 30 to 35 wherein the host cell is a eukaryote or archae cell.

45. The host cell of claims 44 wherein the eukaryote cell is a fungus or a yeast.

46. The host cell of claims 45 wherein the fungus is a filamentous fungus.

47. The host cell of claims 46 wherein the filamentous fungus is of the genus Aspergillus.

48. The host cell of claims 47 wherein the filamentous fungus is of the species oryzae, niger, or aculeatus.

49. The host cell of claims 45 wherein the yeast is of the genus Saccharomyces, Yarrovia or Pichia.

50. The host cell of claims 49 wherein the yeast is of the species P. pastoris, S. Calsbergensis, S. cerevisiae, or Y. lipolytica.

51. The host cell of anyone of claims 30 - 50 wherein the host cell is free of genetic modications causing a stress response from sensed by the biosensor in the absence of production of the compound of interest.

52. The host cell of claim 51 wherein the genetic modications is attenuation, disruption or deletion of a gene encoding a phosphatidylserine synthase.

53. A cell culture comprising the host cell of anyone of claims 30 to 52 and a growth medium.

54. A method for determining cellular stress in a host cell comprising f) introducing the biosensor nucleotide gene construct of anyone of claims 1 to 25 or the vector of anyone of claims 26 to 29 into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; g) cultivating the host cell so as to allow the host cell to perform a metabolic process producing a compound of interest; h) measuring expression of the biomarker; and i) determining from the level of expression of the biomarker if the host cell is stressed from its performance of the metabolic process of interest.

55. A method for monitoring production of a compound of interest in a host cell comprising f) introducing the biosensor nucleotide gene construct of anyone of claims 1 to 25 or the vector ofanyone of claims 26 to 29 into the host cell so that the host cell is capable of expressing the biomarker upon inducing the promoter; g) cultivating the host cell so as to allow it cell to perform a metabolic process producing the compound of interest of interest; h) measuring expression of the biomarker; i) determining at least one parameter of the production of the compound of interest of interest from the level of expression of the biomarker; and j) optionally changing at least one parameter of the metabolic process or the cultivation conditions to change, optionally lower, expression of the biomarker.

56. A method for selecting host cells exhibiting reduced cellular stress comprising: a) introducing the biosensor nucleotide gene construct of anyone of claims 1 to 25 or the vector of anyone of claims 26 to 29 into a host cell performing a metabolic process producing a compound of interest, so that the host cell is capable of expressing the biomarker upon inducing the promoter; b) modifying the host cell by modifying one or more elements of the metabolic pathway of interest to create a library of modified host cells; c) cultivating the library of modified host cell so as to allow it to perform the modified metabolic processes of interest; d) measuring expression of the biomarker in the library of modified host cells upon performing the modified metabolic pathway of interest; and e) selecting modified host cells exhibiting reduced cellular stress by having reduced expression of the biomarker.

57. The methods of anyone of claims 54 to 56 wherein the compond of interest is a protein of peptide, optionally a protein of peptide produced in the periplasm of the host cell.

58. A biosensor kit of parts for introducing a biosensor into a host cell comprising the biosensor nucleotide gene construct of anyone of claims 1 to 25 and / or the vector of anyone of claims 26 to 29 and one or more elements selected from a plasmid, competent cells, gene fragment for integration into a vector, primers, enzymes, and / or buffers, and optionally instructions for use.* * *

Images