Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system.

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl ß-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.

Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. RESULTS: To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. CONCLUSION: These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.

Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

Rice N-biofertilization by inoculation with an engineered photosynthetic diazotroph.

Photosynthetic diazotrophs expressing iron-only (Fe-only) nitrogenase can be developed into a promising biofertilizer, as it is independent on the molybdenum availability in the soil. However, the expression of Fe-only nitrogenase in diazotrophs is repressed by the fixed nitrogen of the soil, limiting the efficiency of nitrogen fixation in farmland with low ammonium concentrations that are inadequate for sustainable crop growth. Here, we succeeded in constitutively expressing the Fe-only nitrogenase even in the presence of ammonium by controlling the transcription of Fe-only nitrogenase gene cluster (anfHDGK) with the transcriptional activator of Mo nitrogenase (NifA*) in several different ways, indicating that the engineered NifA* strains can be used as promising chassis cells for efficient expression of different types of nitrogenases. When applied as a biofertilizer, the engineered Rhodopseudomonas palustris effectively stimulated rice growth, contributing to the reduced use of chemical fertilizer and the development of sustainable agriculture.

Genetic polymorphism, constitutive expression and tissue localization of Dirofilaria immitis P-glycoprotein 11: a putative marker of macrocyclic lactone resistance.

BACKGROUND: Dirofilaria immitis causes dirofilariosis, a potentially fatal condition in canids. Dirofilaria infections can be prevented with a macrocyclic lactone (ML) prophylactic regimen. However, some D. immitis isolates have become resistant to MLs. Genetic changes on the P-glycoprotein 11 gene, encoding an ABCB transporter, have been linked to the ML-resistant phenotypes and have been proposed as markers of drug resistance. However, nothing is known about the expression and the localization of this transporter in D. immitis, despite its strong link to ML-resistant phenotypes. METHODS: We examined the clinically validated D. immitis P-glycoprotein 11 (DimPgp-11) single nucleotide polymorphism (SNP) via MiSeq analysis in three ML-susceptible isolates (Missouri, MP3 and Yazoo) and two ML-resistant isolates (JYD-34 and Metairie), and correlated the data with previously published MiSeq results of USA laboratory-maintained D. immitis isolates. The level of the expression of the DimPgp-11 messenger RNA transcript was analyzed by droplet digital PCR (ddPCR) and compared in the USA laboratory-maintained isolates, namely the ML-susceptible Missouri and Berkeley isolates, the putative ML-susceptible Georgia III and Big Head isolates and the ML-resistant isolate JYD-34. The immunolocalization of DimPgp-11 was visualized in the microfilaria (mf) life stage of the Missouri isolate using confocal microscopy. RESULTS: The results confirmed that the SNP found on DimPgp-11 is differentially expressed in the USA laboratory-maintained isolates. The ML-susceptible isolates had an alternate allele frequency of between 0% and 15%, while it ranged between 17% and 56% in the ML-resistant isolates. The constitutive expression of DimPgp-11 was similar in the Berkeley, Georgia III and Big Head isolates, while it was significantly decreased in the ML-resistant JYD-34 isolate (P < 0.05), when compared to the ML-susceptible Missouri isolate. The DimPgp-11 protein was distinctly localized within the excretory-secretory (ES) duct, pore cells and the excretory cell and, more faintly, along the mf body wall. CONCLUSION: Our data confirm that genetic polymorphism of DimPgp-11 is associated with ML resistance in USA laboratory-maintained D. imminits isolates. A link between DimPgp-11 and ML resistance in D. immitis is further supported by the lower protein expression in the ML-resistant JYD-34 isolate when compared with the ML-susceptible Missouri isolate. Interestingly, DimPgp-11 is strategically located surrounding the ES pore where it could play an active role in ML efflux.

Molecular Characterization of an Isoflavone 2'-Hydroxylase Gene Revealed Positive Insights into Flavonoid Accumulation and Abiotic Stress Tolerance in Safflower.

Flavonoids with significant therapeutic properties play an essential role in plant growth, development, and adaptation to various environments. The biosynthetic pathway of flavonoids has long been studied in plants; however, its regulatory mechanism in safflower largely remains unclear. Here, we carried out comprehensive genome-wide identification and functional characterization of a putative cytochrome P45081E8 gene encoding an isoflavone 2'-hydroxylase from safflower. A total of 15 CtCYP81E genes were identified from the safflower genome. Phylogenetic classification and conserved topology of CtCYP81E gene structures, protein motifs, and cis-elements elucidated crucial insights into plant growth, development, and stress responses. The diverse expression pattern of CtCYP81E genes in four different flowering stages suggested important clues into the regulation of secondary metabolites. Similarly, the variable expression of CtCYP81E8 during multiple flowering stages further highlighted a strong relationship with metabolite accumulation. Furthermore, the orchestrated link between transcriptional regulation of CtCYP81E8 and flavonoid accumulation was further validated in the yellow- and red-type safflower. The spatiotemporal expression of CtCYP81E8 under methyl jasmonate, polyethylene glycol, light, and dark conditions further highlighted its likely significance in abiotic stress adaption. Moreover, the over-expressed transgenic Arabidopsis lines showed enhanced transcript abundance in OE-13 line with approximately eight-fold increased expression. The upregulation of AtCHS, AtF3'H, and AtDFR genes and the detection of several types of flavonoids in the OE-13 transgenic line also provides crucial insights into the potential role of CtCYP81E8 during flavonoid accumulation. Together, our findings shed light on the fundamental role of CtCYP81E8 encoding a putative isoflavone 2'-hydroxylase via constitutive expression during flavonoid biosynthesis.

Characterization of two constitutive promoters RPS28 and EIF1 for studying soybean growth, development, and symbiotic nodule development.

Native promoters that can drive high and stable transgene expression are important tools for modifying plant traits. Although several such promoters have been reported in soybean (Glycine max), few of them function at multiple growth and development stages and during nodule development. Here, we report that the promoters of 40S RIBOSOMAL PROTEIN SMALL SUBUNIT S28 (RPS28) and EUKARYOTIC TRANSLATION INITIATION FACTOR 1 (EIF1) are ideal for high expression of transgene. Through bioinformatic analysis, we determined that RPS28 and EIF1 were highly expressed during soybean growth and development, nodule development, and various biotic and abiotic stresses. Fusion of both RPS28 and EIF1 promoters, with or without their first intron, with the reporter gene ß-GLUCURONIDASE (uidA) in transgenic soybean, resulted in high GUS activity in seedlings, seeds, and nodules. Fluorimetric GUS assays showed that the RPS28 promoter and the EIF1 promoter yielded high expression, comparable to the soybean Ubiquitin (GmUbi) promoter. RPS28 and EIF1 promoters were also highly expressed in Arabidopsis thaliana and Nicotiana benthamiana. Our results indicate the potential of RPS28 and EIF1 promoters to facilitate future genetic engineering and breeding to improve the quality and yield of soybean, as well as in a wide variety of other plant species. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00073-6.

Construction of constitutive expression of Eimeria tenella eukaryotic initiation factor U6L5H2 on the surface of Lactobacillus plantarum and evaluation of its immunoprotective efficiency against chicken coccidiosis.

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens.

A new platform host for strong expression under GAL promoters without inducer in Saccharomyces cerevisiae.

The gal80 mutant of yeast Saccharomyces cerevisiae is used for the constitutive expression under strong GAL promoters without galactose induction. To enhance productivity of gal80 mutant, an alternative strain, allgal, was developed by removing all galactose-utilizing genes that consume significant cellular resources in the gal80 strain when cultured in non-galactose conditions. The efficacy of the allgal mutant (gal80, gal1, gal2, gal7, and gal10) was verified by assessing the secretory expression of three recombinant proteins, Candida antarctica lipase B (CalB), human serum albumin (HSA), and human epidermal growth factor (hEGF), using the GAL10 promoter. The growth of the allgal mutant was enhanced by 15-38% compared to the gal80 mutant, and the secretion of recombinant proteins also increased by 16-22% in fed-batch fermentation. Thus, the expression of recombinant proteins using GAL10 promoter in the allgal mutant is suitable for the economical production of recombinant proteins in S. cerevisiae.

Characterization of an N-acylhomoserine lactonase from Serratia sp. and its biofouling mitigation in a membrane bioreactor.

Membrane biofouling is a process that can impede the development of membrane bioreactor (MBR), which constitutes an important system of the wastewater treatment process. Membrane biofouling is governed by quorum sensing (QS), a communication system heavily dependent on the activities of signal molecules. Certain bacteria, known as quorum quenching (QQ) bacteria, can quench the QS process by destroying the signal molecules. These QQ bacteria are considered a sustainable and feasible way of mitigating membrane biofouling in MBR. In this study, a QQ enzyme (designated as AisZ) from a Serratia sp. was first identified and characterized. Escherichia coli BL21 expressing AisZ was able to degrade different QS signal molecules. Furthermore, these cells could also mitigate membrane biofouling in MBR during a 29-day operation by reducing the transmembrane pressure from 31 to 21 kPa. The metal ions Co2+ and Ni2+ were relatively important to AisZ in that they could significantly enhance the activity of AisZ and restore the EDTA-inactivated AisZ. Expression of the aisA gene was not influenced by Co2+, Ni2+ and QS signal molecules. AisZ might, therefore, extend the diversity of potential candidates for the mitigation of biofouling associated with membrane filtration technologies.

Plasmid-Based Gene Expression Systems for Lactic Acid Bacteria: A Review.

Lactic acid bacteria (LAB) play a very vital role in food production, preservation, and as probiotic agents. Some of these species can colonize and survive longer in the gastrointestinal tract (GIT), where their presence is crucially helpful to promote human health. LAB has also been used as a safe and efficient incubator to produce proteins of interest. With the advent of genetic engineering, recombinant LAB have been effectively employed as vectors for delivering therapeutic molecules to mucosal tissues of the oral, nasal, and vaginal tracks and for shuttling therapeutics for diabetes, cancer, viral infections, and several gastrointestinal infections. The most important tool needed to develop genetically engineered LABs to produce proteins of interest is a plasmid-based gene expression system. To date, a handful of constitutive and inducible vectors for LAB have been developed, but their limited availability, host specificity, instability, and low carrying capacity have narrowed their spectrum of applications. The current review discusses the plasmid-based vectors that have been developed so far for LAB; their functionality, potency, and constraints; and further highlights the need for a new, more stable, and effective gene expression platform for LAB.

Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast.

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

ERAS Is Constitutively Expressed in the Tissues of Adult Horses and May Be a Key Player in Basal Autophagy.

ERas is a new gene of the Ras family found in murine embryonic stem (ES) cells. Its human ortholog is not expressed in human ES cells. So far ERas gene has only been found to be expressed in the tissues of adult cynomolgus monkeys and cattle; however, information about ERAS expression or its potential functions in equine tissues is lacking. This study was performed to investigate whether Eras is an equine functional gene and whether ERAS is expressed in the tissues of adult horses and determine its potential physiological role. Expression of the ERas gene was detected in all examined adult tissues, and the RT-PCR assay revealed ERAS transcripts. Protein expression was also detected by Western blot analysis. Quantitative real time RT-qPCR analysis revealed that different expression levels of ERAS transcripts were most highly expressed in the testis. Immunohistochemically, ERAS was found to be localized prevalently in the plasmatic membrane as well as cytoplasm of the cells. ERAS was a physical partner of activated PDGFßR leading to the AKT signaling. ERAS was found to interact with a network of proteins (BAG3, CHIP, Hsc70/Hsp70, HspB8, Synpo2, and p62) known to play a role in the chaperone-assisted selective autophagy (CASA), which is also known as BAG3-mediated selective macroautophagy, an adaptive mechanism to maintain cellular homeostasis. Furthermore, ERAS was found to interact with parkin. PINK1, BNIP3, laforin. All these proteins are known to play a role in parkin-dependent and -independent mitophagy. This is the first study demonstrating that Eras is a functional gene, and that ERAS is constitutively expressed in the tissues of adult horses. ERAS appears to play a physiological role in cellular proteostasis maintenance, thus mitigating the proteotoxicity of accumulated misfolded proteins and contributing to protection against disease. Finally, it is conceivable that activation of AKT pathway by PDGFRs promotes actin reorganization, directed cell movements, stimulation of cell growth.

Constitutive expression of nitrilase from Rhodococcus zopfii for efficient biosynthesis of 2-chloronicotinic acid.

2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from Rhodococcus zopfii (RzNIT/W167G) was constitutively overexpressed with Escherichia coli as host, which exhibited a onefold increase in enzymatic activity compared with inducible expression. Biosynthesis of 2-CA using whole cells harboring nitrilase as biocatalysts were investigated and 318.5 mM 2-CA was produced, which was the highest level for 2-CA production catalyzed by nitrilase to date. 2-CA was recovered from the reaction mixture through a simple acidification step with a recovery yield of 90%. This study developed an efficient bioprocess for 2-CA with great potential for industrial application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03119-0.

Whole-cell biocatalyst for cadaverine production using stable, constitutive and high expression of lysine decarboxylase in recombinant Escherichia coli W3110.

Microbial production of industrial chemicals is a sustainable approach to reduce the dependence on petroleum-based chemicals such as acids, alcohols, and amines, in which the cadaverine is a natural diamide and serves as one of the key monomers for biopolymer production. In this study, the constitutive promoter J23100 driven lysine decarboxylase (CadA) for cadaverine production was established and compared in different Escherichia coli strains. The best chassis designed as JW, expressed the highest amount of CadA by using J23100 promoter, showing stable and high copy numbers (i.e., PCN > 100) when culture in the antibiotic-free medium. JW attained a CadA activity of 167 g-DAP/g-DCW-h and had the maximum biocatalyst of 45.6 g-DCW/L in fed-batch fermentation. In addition, JW was able to convert 2.5 M L-lysine to 221 g/L cadaverine, with 86 % yield and 55.3 g/L-h productivity. The whole-cell biocatalyst could be reused over four times at an average of 97 % conversion when supplied half of fresh cells in the reaction. This work developed a stable, constitutive expression, long-term preservation, high-level expression of CadA for DAP production, and paved an alternative opportunity of bio-nylon for industry in the future.

Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris.

Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45-50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications.

Efficient Constitutive Expression of Cellulolytic Enzymes in Penicillium oxalicum for Improved Efficiency of Lignocellulose Degradation.

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δerm.

The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.

Engineering a Synthesis-Friendly Constitutive Promoter for Mammalian Cell Expression.

Since cell-based therapies require the constitutive and stable expression of therapeutic transgenes, lentiviral infection is commonly used to integrate gene material regulated by standard constitutive promoters. Unfortunately, none of the standard or synthetic constitutive promoters can be easily synthesized at low cost due to the presence of repeated subsequences. Thus, in this paper, we designed a synthetic constitutive promoter (named SFCp) that can drive the expression of fluorescent proteins that subsequently trafficked to intended subcellular localizations and the expression of synthetic proteins that rewired the cellular response of Ca2+ to cell morphology changes. Furthermore, SFCp can be used to avoid sequence homology that can theoretically result in loss of genetic material by homologous recombination in tandem constructs. As gene synthesis becomes an indispensable tool in the arsenal of synthetic biology, it is essential to develop a toolbox of gene synthesis friendly components for cell engineering such as constitutive promoters.

High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris.

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105 U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105 U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105 U/mL. Finally, LHyal activity of 2.12 × 106 U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104 U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.