Protein Production In Bacteria Research Articles

Botryococcus braunii autolysate for the production of deuterium-labeled recombinant protein

Deuterated biomolecules such as proteins, lipids, and DNA are widely used in neutron scattering experiments. This is due to the unique scattering properties of 2H, including a strong positive neutron scattering length while contributing very little background compared to the more abundant 1H isotope. Deuteration is therefore an indispensable component in the study of structure, function, and dynamic behaviour of biomolecules by neutron scattering. In the past we compared multiple microalgae species for their ability to grow under deuterated conditions and in our hands Botryococcus braunii proved the easiest and most resilient to long-term culturing in D2O. In this study we describe how to culture B. braunii cells under deuterated conditions followed by preparation of an aqueous extract. The procedure is based on autolysis where cells are incubated at 50 °C for 24 h and clarified by centrifugation and filtration. The product, deuterated algal autolysate, is then used in minimal media for deuterated recombinant protein production in bacteria. We demonstrate that in-house produced deuterated algal autolysate can fully substitute for glycerol-d8 in minimal media without a reduction in expressed protein yield while obtaining ~98 % deuterium incorporation in the final product, suitable for neutron scattering and other types of experiments.

Multimodal Control of Bacterial Gene Expression by Red and Blue Light.

By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

Predicting stress response and improved protein overproduction in Bacillus subtilis

Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis, iJT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed iJT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like iJT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

Quality comparison of recombinant soluble proteins and proteins solubilized from bacterial inclusion bodies

Recombinant protein production in bacteria is often accompanied by the formation of aggregates, known as inclusion bodies (IBs). Although several strategies have been developed to minimize protein aggregation, many heterologous proteins are produced in aggregated form. For these proteins, purification necessarily requires processes of solubilization and refolding, often involving denaturing agents. However, the presence of biologically active recombinant proteins forming IBs has driven a redefinition of the protocols used to obtain soluble protein avoiding the protein denaturation step. Among the different strategies described, the detergent n-lauroylsarcosine (NLS) has proved to be effective. However, the impact of the NLS on final protein quality has not been evaluated so far. Here, the activity of three antimicrobial proteins (all as GFP fusions) obtained from the soluble fraction was compared with those solubilized from IBs. Results showed that NLS solubilized proteins from IBs efficiently, but that protein activity was impaired. Thus, a solubilization protocol without detergents was evaluated, demonstrating that this strategy efficiently solubilized proteins embedded in IBs while retaining their biological activity. These results showed that the protocol used for IB solubilization has an impact on final protein quality and that IBs can be solubilized through a very simple step, obtaining fully active proteins.

Selection of High Producers From Combinatorial Libraries for the Production of Recombinant Proteins in Escherichia coli and Vibrio natriegens.

The optimization of recombinant protein production in bacteria is an important stage of process development, especially for difficult-to-express proteins that are particularly sensitive or recalcitrant. The optimal expression level must be neither too low, which would limit yields, nor too high, which would promote the formation of insoluble inclusion bodies. Expression can be optimized by testing different combinations of elements such as ribosome binding sites and N-terminal affinity tags, but the rate of protein synthesis is strongly dependent on mRNA secondary structures so the combined effects of these elements must be taken into account. This substantially increases the complexity of high-throughput expression screening. To address this limitation, we generated libraries of constructs systematically combining different ribosome binding sites, N-terminal affinity tags, and periplasmic translocation sequences representing two secretion pathways. Each construct also contained a green fluorescent protein (GFP) tag to allow the identification of high producers and a thrombin cleavage site enabling the removal of fusion tags. To achieve proof of principle, we generated libraries of 200 different combinations of elements for the expression of an antimicrobial peptide (AMPs), an antifungal peptide, and the enzyme urate oxidase (uricase) in Escherichia coli and Vibrio natriegens. High producers for all three difficult-to-express products were enriched by fluorescence-activated cell sorting. Our results indicated that the E. coli ssYahJ secretion signal is recognized in V. natriegens and efficiently mediates translocation to the periplasm. Our combinatorial library approach therefore allows the cross-species direct selection of high-producer clones for difficult-to-express proteins by systematically evaluating the combined impact of multiple construct elements.

Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production.

The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here, we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give a controlled expression that is suitable for diverse biotechnology applications.

The new strategies to overcome challenges in protein production in bacteria.

Recombinant proteins are essential for biotechnology. Here we review some of the key points for improving the production of heterologous proteins, and what can be the future of the field.

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

BackgroundThe industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential.ResultsIn this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed.ConclusionsThe total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described.Graphical abstractAntibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

CRISPRi-Manipulation of Genetic Code Expansion via RF1 for Reassignment of Amber Codon in Bacteria

The precise engineering of proteins in bacteria via the amber codon has been hampered by the poor incorporation of unnatural amino acid (UAA). Here we explored the amber assignment as a sense codon for UAA by CRISPRi targeting release factor 1 (RF1). Scanning of RF1 gene with sgRNAs identified target loci that differentiate RF1 repressions. Quantitation of RF1 repressions versus UAA incorporation indicated an increasing interrelation with the amber reassignment maximized upon RF1 knockdown to ~30%, disclosing the beneficial role of RF1 in amber assignment. However, further RF1 repression reversed this trend resulting from the detrimental effects on host cell growth, disclosing the harmful aspect of RF1 in reassignment of the amber codon. Our data indicate RF1 as a switch manipulating genetic code expansion and pave a direction via CRISPRi for precise engineering and efficient production of proteins in bacteria.

Thioflavin-S Staining of Bacterial Inclusion Bodies for the Fast, Simple, and Inexpensive Screening of Amyloid Aggregation Inhibitors

Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer's disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer's related β-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD.

Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen Acinetobacter baumannii

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.

Next-generation sequencing of the CHO cell transcriptome

Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro cultivation as they require assimilable low sustenance [1]. Today, CHO cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins and are considered as the mammalian equivalent of E. coli in research and biotechnology [2]. The production of biopharmaceuticals in CHO cells is superior to protein production in bacteria, because mammalian cell lines procure complex folding and post-translational modifications like glycosylation. However, contrary to the increasing importance in biotechnology and industry, comprehensive genome and transcriptome information of CHO cell lines is still rare. In this study, the pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences were used to achieve a major step forward towards unraveling the transcriptome of CHO cells. CHO cDNA samples derived from different CHO cell lines and growth conditions were used for the generation of 1.84 mill. high quality sequencing reads with an average read length of 373 nt summing up to 603 Mb data. Assembly of the sequencing data resulted in 41,039 contiguous sequences. These contigs were grouped by the Newbler software into 36,383 isotigs and 28,039 isogroups. Taxonomical classification and comparison to the Mus musculus transcriptome demonstrated the actual quality of the CHO cell line sequences. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway and the central metabolism of CHO cells were detected. Only fructose-1,6-bisphosphatase (3.1.3.11) and 6-phosphogluconolactonase (3.1.1.31) were not identified within the pentose phosphate pathway. The newly sequenced CHO cell line transcriptome was the basis for the design of a customized CHO microarray. Contig sequences were used for the design of 94.580 probes. The designed probes cover 31,905 splice variants of CHO transcripts. With a Self-Self Hybridization experiment (Figure ​(Figure1)1) the functionality of the probes was demonstrated. This experiment was performed with the same RNA as which was used for sequencing. Half of this RNA was labeled with Cy3, the other half with Cy5. For this study the dye intensity, the dye ratio and the adjusted p-value (student´s t-test, FDR controlled, α=0.05) of four microarray replicates were analyzed. It is expected, that the two labeled RNA samples bind equally to the probe, if a transcript is expressed (Figure ​(Figure1:1: pink, dark blue, red, light blue). Only the probes for one transcript could be rejected, because of their dysfunctionality (Figure ​(Figure1,1, green). Figure 1 Self-Self Hybridization experiment of the customized CHO microarray. The dye ratio (M-value) is plotted against the adjusted p-value (student´s t-test, FDR controlled, α=0.05). The dye ratio was calculated using the mean intensity values ... This CHO microarray is now available for further experiments and will support transcriptional analysis of CHO cells under process conditions for cell line and process optimization. It was used already used successfully for a gene expression study of CHO DP-12 cells cultivated under sodium butyrate treatment [3].

SPY-ing into Protein Stability

Production of human proteins in bacteria has always been an arduous task, as has also been realized by biochemists while working with proteins. We expect our protein to be secreted into surrounding medium instead its seen as insoluble inclusion body in precipitated form inside the bacteria or is found glued to the cell wall of bacteria and may sometimes be, unfortunately seen as lumps at the bottom of test tubes. Keeping this problem in mind, the primary goal of protein engineering, rational drug design and biopharmaceutical production is the development, production and storage of stable proteins with full functionality. In in vivo systems, chaperones are the proteins responsible for folding of proteins into their biologically active conformations and preventing their aggregation. So, the key here is to search for a molecule; which can stabilize the correctly folded structure or a drug that can disrupt pathways leading to protein misfolding. This has really come true. A research team led by Prof. James Bardwell, University of Michigan spearheaded the discovery of molecular chaperone protein Spy in E. coli. When E. coli was challenged to stabilize a very unstable periplasmic mutant protein Im7, it massively overproduced another periplasmic protein Spy thereby, increasing the steady state levels of Im7 by 700 folds [1, 2]. In vitro studies demonstrate that Spy, a 15.9 KDa protein, is an effective ATP-independent chaperone that causes suppression of protein aggregation. Unlike many other ATP independent chaperones, e.g. DnaK and GroeL systems, it also helps in protein refolding [3]. This study is supported by the effects of Spy in preventing Malate dehydrogenase aggregation due to denaturation by urea. Similar results have been seen in prevention of tannin-mediated inactivation of E. coli membrane protein by Spy where it protects the membrane proteins from tannic acid-induced activity loss, and interestingly it worked as a chaperone even in substoichiometric quantities, suggesting its high efficiency. Its expression is under the control of Bae and Cpx periplasmic stress response systems which are induced by a variety of stress conditions known to cause protein unfolding and aggregation, e.g. heat, urea, ethanol, butanol, all of which use different mechanisms to unfold proteins [4, 5]. This suggests that Spy has a general affinity for a wide range of different protein-unfolding intermediates. This general affinity to shield the aggregation-sensitive regions of a variety of protein-unfolding intermediates has been attributed to its dimer formation with a novel α helix and absence of globular core which allows higher flexibility. These properties of Spy can thus be exploited to manufacture proteins to be used in medicines, industry as well as research. Spy thus, appears to be one of the very few chaperones that actively support protein refolding in absence of any obvious energy source and other regulatory proteins suggesting that Spy uses a mechanism to control substrate binding and release that is different from any of the previously characterized chaperones; although how it accomplishes it, is a prospective area of future research.

Recombinant protein expression system in cold loving microorganisms

Background Soluble and functional proteins are of high demand in modern biotechnology. Although many recombinant proteins have been successfully obtained from common prokaryotic and eukaryotic hosts, these systems result to be often unproductive due to the peculiar properties of the protein to be produced. Incorrect folding of the nascent polypeptide chains is one of the main problems occurring during heterologous protein production in bacteria. Since formation of inclusion bodies often impairs the recombinant production of valuable proteins, many experimental approaches have been explored to minimize this undesirable effect [1,2]. Expression of difficult proteins has also been carried out by lowering the temperature at the physiological limit allowed for the growth of mesophilic host organisms (between 15 and 18°C for Escherichia coli). Lowering the temperature, in fact, has a pleiotropic effect on the folding process, destabilising the hydrophobic interactions needed for intermediates aggregation [3]. On the basis of the above considerations, a rational alternative to mesophilic organisms is the use of naturally cold-adapted bacteria as hosts for protein production at low temperature (even at around 0°C).

Sensor combination and chemometric modelling for improved process monitoring in recombinant E. coli fed-batch cultivations

The key objective for the optimisation of recombinant protein production in bacteria is to optimize the exploitation of the host cell's synthesis potential. Recent studies show that the novel concept of transcription rate control allows the tuning of recombinant gene expression in relation to the metabolic capacity of the host cell. To adjust the inducer–biomass ratio to a tolerable level, real-time knowledge about key process variables is paramount. Since there are no reliable online-sensors for key variables such as biomass or recombinant product, it is necessary to relate available online signals to process variables by mathematical models. To improve chemometric modelling of process variables, dielectric spectroscopy and a multi-wavelength online fluorescence sensor for two-dimensional fluorescence spectroscopy were applied in a series of recombinant Escherichia coli fed-batch cultivations applying two different process operation states. Dielectric spectroscopy signals were closely correlated to biomass, while two-dimensional fluorescence spectroscopy allowed the monitoring of fluorescent biogenic components. Chemometric modelling of key process variables with two different modelling techniques showed that this sensor combination greatly improved the estimation (i.e. reduce error magnitude) of process variables in recombinant E. coli cultivations, thereby enhancing process monitoring capabilities.

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies.

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between "yield" and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially "screen," in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts.

Stabilizing plasmid copy number to improve recombinant protein production.

The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.

HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in differentH. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno–electron microscopy (IEM), and phase–contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3–4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription–polymerase chain reaction (RT–PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.

Synthesis of proteins in Escherichia coli immunoreactive with sera from individuals infected with human T-cell leukemia virus type I.

The env-pX IV fused gene of human T-cell leukemia virus type I (HTLV-I) was inserted into lac promoter-directed expression vectors for production of viral proteins in bacteria. Resulting recombinant plasmids, pK13 and pK15, directed synthesis of fused proteins of 59 kDa (Env-p40x) and 100 kDa (Gag-Env-p40x), respectively. Western blot analysis showed that these proteins were reactive with sera of patients with adult T-cell leukemia (ATL) and retained multiple antigenic determinants of viral proteins. In combination with recombinant Gag protein [S. Itamura, K. Shigesada, M. Imai, N. Kobayashi, T. Hamakado, T. Harada and M. Hatanaka, Gene 38, 57-64 (1985)], these bacterially synthesized proteins may provide a useful tool for differential diagnosis of ATL by detecting serum antibodies against individual viral proteins and for analysis of viral gene functions.

Population differences in body size, shape and growth rate. A 1976 view.

<h3>Abstract</h3> Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells, purified, and resolubilized. The <i>Salmonella</i> pathogenicity island 1 type III secretion system (T3SS) is a protein secretion complex in <i>Salmonella</i> that has been engineered to secrete heterologous proteins and addresses the problems associated with bacterial protein production. However, the current best practices method of T3SS pathway activation for secretion is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, <i>hilA</i>, which requires the addition of a small molecule inducer to the culture media and adds significant cost to the production media. Plasmid-based expression is also subject to instability in large-scale fermentation. Here, we show that we can constitutively activate the T3SS by modulating the upstream transcriptional regulator, <i>hilD</i>, either through knocking out <i>hilE</i>, a repressor of HilD, or by adding transcriptional fusions to <i>hilD</i>. Finally, we combine the two most promising genomic modifications to build a constitutively active T3SS capable of secreting a range of heterologous proteins at titers comparable to those reported with synthetic induction of <i>hilA</i>. These improvements further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. <h3>Importance</h3> Proteins are used in our everyday lives as therapeutics (insulin), industrial enzymes (laundry detergent), and bio-based materials (spider silk). Current industrial protein production in bacteria is costly because it requires purification of the target protein from the other proteins inside the cell. We solve this problem by engineering the <i>Salmonella</i> pathogenicity island 1 type III secretion system (T3SS) to export the target protein into the cell growth media. This makes the protein purification process more efficient and cheaper. However, this system currently requires an expensive inducer reagent to activate it which significantly increases the cost of the production media. We show here the creation of a constitutively active T3SS, meaning the T3SS pathway is always on. In doing so, we successfully created a <i>Salmonella</i> strain that eliminates the need for the inducer reagent and exports proteins at levels comparable to the inducer-activated system, ultimately reducing the cost of T3SS protein production.