Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation

The application of safe and cheap inducers is important in the field of fermentation technology, which persuades employing new expression systems. In this study, a Bacillus subtilis expression system was induced by applying starvation and environmental stresses to produce xylanase. The expression plasmid harbors SigB-dependent ohrB promoter. The target gene was expressed by inoculating the recombinant strain into glucose-limited synthetic medium resulting in a sharp increase of xylanase activity at the end of logarithmic growth phase. The recombinant strain was able to express the xylanase enzyme 14-fold higher than that of the control one. The induction was also performed by exposing the recombinant strain to NaCl and ethanol stresses, and heat shock; the strain growing in LB showed 5-, 15- and 6-fold increases in xylanase activity, respectively. The best induction using environmental stresses was achieved by applying the salt stress in the synthetic medium. The maximum expression for NaCl and ethanol stresses occurred after 40 min of induction. All observed inductions were related to activation of SigB protein causing expression of the SigB-dependent xylanase gene. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ‘‘one amino acid-one codon”-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.     

Soluble expression of pullulanase from Bacillus acidopullulyticus in Escherichia coli by tightly controlling basal expression

Bacillus acidopullulyticus pullulanase (BaPul13A) is a widely used debranching enzyme in the starch industry. A few details have been reported on the heterologous expression of BaPul13A in Escherichia coli (E. coli). This study compares different E. coli expression systems to improve the soluble expression level of BaPul13A. When pET22b(+)/pET28a(+) was used as the expression vector, the soluble expression of BaPul13A can be achieved by tightly controlling basal expression, whereas pET-20b(+)/pGEX4T2 leads to insoluble inclusion bodies. An efficient process control strategy aimed at minimizing the formation of inclusion bodies and enhancing the production of pullulanase was developed by a step decrease of the temperature in a 5-L fermentor. The highest total enzyme activity of BaPul13A reached 1,156.32 U/mL. This work reveals that the T7 promoter with lac operator and lacI gene collectively contribute to the soluble expression of BaPul13A, whereas either a T7 promoter alone or combined with the lac operator and lacI gene results in poor solubility. Basal expression in the initial growth phase of the host significantly affects the solubility of BaPul13A in E. coli.     

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L^?1 and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL^?1 was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL^?1) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L^?1 glucose led to a 6.2-fold increase (465.07 U mL^?1) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.     

Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy

Halohydrin dehalogenases are attractive biocatalysts in producing a series of important chiral building blocks. Recombinant expression of halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) in Escherichia coli using T7 promoter-based pGEF(+) system revealed much lower expression level than that of the well-studied halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). In this study, we changed the codon usage in the 5′-end of hheA gene to improve the expression yield of HheA. Our results showed that the expression of HheA could be largely improved by the replacement of G-rich +2 codon (adjacent to the start codon) with less G-containing codons. The expression of one of the resulting mutants HheA-D1 (replaced +2 codon GTG with CCA) was about 4-fold higher and purified yields about 8-fold greater than that of the wild-type HheA. Moreover, the expression level of the resulting HheA variants correlated well with the minimal folding free energy (ΔG) of the mRNA secondary structure surrounding the 5′-end region of the genes. These findings suggested that the G-rich +2 codon of hheA gene might be the main suppressive factor for limiting the recombinant expression of HheA and that +2 codon optimization strategy could be used as a general tool in modulating recombinant protein production in E. coli.     

pHsh vectors,a novel expression system of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the large-scale production of recombinant enzymes

Gene expression system Hsh is developed to increase enzyme production and to decrease the cost in the induction of gene expression in Escherichia coli. The vectors of Hsh system were constructed by combining a synthesized heat-shock promoter with a synthesized terminator and an origin of replication derived from pUC19 in which the expression of foreign genes was regulated by an alternative sigma factor, σ³² of E. coli. In comparison, the Hsh promoter gave a 2.4-fold higher production for xynIII gene encoding a xylanase than existing heat-shock inducible promoter p _L, 1.2-fold and 3-fold production for xar gene encoding a arabinosidase than trc and T₇ promoter, respectively. The flow-in-heat technique created a rapid rise in temperature for effective induction of gene expression in bioreactor scale.     

A novel xylanase from Streptomyces sp. FA1: Purification,characterization, identification,and heterologous expression

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ～ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA ^48-436 , was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.     

Characterization, cloning and functional expression of novel xylanase from Thermomyces lanuginosus SS-8 isolated from self-heating plant wreckage material

Extracellular cellulase free xylanase from Thermomyces lanuginosus sp. SS-8, isolated from self heating plant wreckage material was identified as β-1,4-endo-xylanase precursor, a monomer of 21.3 kDa with no carbohydrate residue. This xylanase retained 80 % activity at 60 °C for 96 h, was active at a wide pH range of 3–11 and uniquely hydrolyzed xylan to xylose without production of xylo-oligosaccharides. Gene xynSS8 encoding xylanase from T. lanuginosus SS-8 was cloned and functionally expressed in Escherichia coli XL1 Blue using pTZ57R/T plasmid and xynSS8/pQE-9 expression vector construct respectively. Gene xynSS8 was of 777 bp and deduced amino acid sequence was a mature xylanase of 258 amino acids. XynSS8 has extra 33 amino acids compared to its nearest homolog and was thermo-alkali tolerant as that of native protein. The xylanase could degrade pulp and release substantial chromophoric materials and lignin derived compounds indicating its effective utility in pulp bleaching. Novel characteristics of the enzyme may contribute to its wide industrial usage. This is first report of cloning and functional expression of the novel xylanase from T. lanuginosus SS-8.     

Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase

A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000–20,000 clones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 310–314. Received 18 May 2000/ Accepted in revised form 19 September 2000     

High secretory production of an alkaliphilic actinomycete xylanase and functional roles of some important residues

Using native signal peptide, an alkaliphilic actinomycete xylanase XynK was overexpressed in Escherichia coli and secreted into the culture medium completely. At its optimum catalytic temperature of 55 °C, the cellulose-free xylanase exhibits high activity and stability at pH 7.0–11.0. In comparison with the well-studied actinomycete xylanase from Streptomyces lividans, as an alkaliphilic xylanase, XynK exhibited different biochemical and catalytic characteristics. With the aid of site-directed mutagenesis, some residues were demonstrated to be important to the activity, stability, or substrate binding of the enzyme. The pH stability of mutants H131S and W135A both decreased obviously under high pH values. Combined with their K _m parameters and homology model analysis, His131 was proposed to be important to both substrate binding and enzyme catalyzing, whereas Trp135 significantly influenced enzyme stability. Good stability under alkaline condition, as well as high secretory expression implies good potentials of the alkaline xylanase in various industrial applications. In addition, results from site-directed mutagenesis provide useful information for further pH stability mechanisms investigation.     

Cloning and expression in Escherichia coli of the glutamate dehydrogenase gene,gdh, from Corynebacterium melassecola

A hybrid plasmid containing a fragment of the Corynebacterium melassecola chromosome cloned into pBR325 restored growth of glutamate auxotrophs of Escherichia coli strains that have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 3.1-kilobase pair region was shown by complementation analysis and enzyme measurements to carry the glutamate dehydrogenase gene, gdh. Glutamate dehydrogenase encoded by gdh carried on recombinant plasmids was elevated over 100-fold in E. coli cells. The gdh promoter was located by in vitro fusion to a promoter-deficient galK gene.     

Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.     

A new strategy for full-length Ebola virus glycoprotein expression in E.coli

Ebola virus(EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein(GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses.However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 ℃. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.     

Improving aquaporin Z expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> by fusion partners and subsequent condition optimization

Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.