Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3).

Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol™ extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 µg/mL after the production and purification of different dsRNAs, reducing production costs by 72%.

Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22.

Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni-NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris-HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters K m, V max, and K cat were 3.06E-05 µg mL-1, 3.06E + 01 µM mg-1 min-1 and 3.27E + 04 s-1, respectively, with EGC as the substrate and 7.14E-07 µg mL-1, 7.14E + 01 µM mg-1 min-1 and 1.40E + 06 s-1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis-Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03073-3.

High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

High-level soluble expression of human Cu,Zn superoxide dismutase with high activity in Escherichia coli.

As the most important member of antioxidant defense system, human Cu,Zn superoxide dismutase (hCu,Zn-SOD) protects cells against the free radicals produced by aerobic metabolism. hCu,Zn-SOD has been widely used in food, cosmetic and medicine industry due to its health benefits and therapeutic potentials. However, a more extensive application of hCu,Zn-SOD is limited by the challenge of expensive and low production of high-activity hCu,Zn-SOD in large scale. In this study, the codon-optimized hCu,Zn-SOD gene was synthesized, cloned into pET-28a( +) and transformed into Escherichia coli BL21(DE3). After induction with IPTG or lactose, hCu,Zn-SOD was highly expressed as soluble form in LB medium with 800 µM Cu2+ and 20 µM Zn2+ at 25 °C. The recombinant hCu,Zn-SOD was efficiently purified by nickel affinity chromatography. Through optimization of fed-batch fermentation conditions, 342 mg purified hCu,Zn-SOD was obtained from 1 L cultures fermented in a 3-L bioreactor. Furthermore, the recombinant hCu,Zn-SOD retained the enzymatic specific activity of 46,541 U/mg. This study has opened up an effective avenue for industrial production of hCu,Zn-SOD through microbial fermentation in the future.

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System.

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Characterization of a lactose-responsive promoter of ATP-binding cassette (ABC) transporter gene from Lactobacillus acidophilus 05-172.

A novel lactose-responsive promoter of the ATP-binding cassette (ABC) transporter gene Lba1680 of Lactobacillus acidophilus strain 05-172 isolated from a traditionally fermented dairy product koumiss was characterized. In L. acidophilus 05-172, expression of Lba1680 was induced by lactose, with lactose-induced transcription of Lba1680 being 6.1-fold higher than that induced by glucose. This is in contrast to L. acidophilus NCFM, a strain isolated from human feces, in which expression of Lba1680 and Lba1679 is induced by glucose. Both gene expression and enzyme activity assays in L. paracasei transformed with a vector containing the inducible Lba1680 promoter (PLba1680) of strain 05-172 and a heme-dependent catalase gene as reporter confirmed that PLba1680 is specifically induced by lactose. Its regulatory expression could not be repressed by glucose, and was independent of cAMP receptor protein. This lactose-responsive promoter might be used in the expression of functional genes in L. paracasei incorporated into a lactose-rich environment, such as dairy products.

How to Determine Interdependencies of Glucose and Lactose Uptake Rates for Heterologous Protein Production with E. coli.

Induction by lactose is known to have a beneficial effect on the expression of soluble recombinant proteins in E. coli harboring the T7 expression system (e.g., E. coli BL21(DE3)). As lactose is a metabolizable inducer, it needs to be supplied continuously to prevent depletion and thus only partial induction. Overfeeding and accumulation of lactose or glucose on the other hand can lead to osmotic stress. Thus, it is of utmost importance to know the possible feeding ranges. Here, we show a fast method using a simple mechanistic model to characterize E. coli strains harboring the T7 expression system regarding their ability to take up lactose and glucose. This approach reduces experimental work and the gained data allows running a stable and robust bioprocess without accumulation of lactose or glucose.

The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl ß-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-ß-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies.

In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a+GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25°C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 UL(-1)h(-1) and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406±521U/L, which was the highest report at fermenter level.