Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.

OBJECTIVE: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins. RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures. CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.

Accumulation Characteristics and Pollution Evaluation of Soil Heavy Metals in Different Land Use Types: Study on the Whole Region of Tianjin.

Heavy metal pollution in soil has received much attention in recent decades. Many studies have analyzed the interaction between specific soil quality and soil heavy metal pollution. However, there is little information about the pollution status, spatial distribution and pollution sources of heavy metals in the province of Tianjin. In this paper, the distribution characteristics and pollution sources of heavy metals in soil were studied by means of the surface soil of Tianjin, as the study area and object, conducted in combination with land use types, using multiple data analysis and multivariate statistics, while the pollution levels were evaluated by various indices. The results showed the mean contents of the seven heavy metals of the studied elements followed an increasing order of Cd (0.15 mg/kg) < As (11.9 mg/kg) < Cu (24.3 mg/kg) = Pb (24.3 mg/kg) < Ni (27.9 mg/kg) < Cr (70.7 mg/kg) < Zn (79.1 mg/kg). The median values of Cr and Ni were lower than the background values and did not exceed the screening values at the points, and the median values of Cu, Zn and Pb were close to the background values, while the median contents of As and Cd were higher than the background values. The highest accumulation of heavy metals was found in grassland, and the coefficient of variation of heavy metal contents were higher in garden land, industrial and mining storage land, residential land and transportation land, indicating that the soil heavy metal contents under these land use types were more significantly disturbed by human factors. The evaluation results of the ground accumulation index method showed that the soil in Tianjin was free of pollution, except for Cd, which was at the non-polluted to moderately polluted level. The Nemero integrated pollution index evaluation method and the pollution load index evaluation method together showed that the integrated pollution levels of heavy metals in Tianjin soils were both at no pollution level/safety level. Apart from Cd and As, which were not correlated, the other heavy metals were correlated with each other two by two. Cd, Pb and Zn were the main pollution contributors from traffic, industry and other anthropogenic factors, while Cr and Ni were the main pollution contributors from soil parent material, and Cu was the main pollution contributor from mining and metal smelting. In addition, As was presumed to be the main source of pollution contribution from agriculture and surface runoff.

A modified method of gene disruption in Komagataella phaffii with Cre/loxP system.

To increase protein production, technologies of gene manipulation for engineering the yeast Komagataella phaffii are extensively exploited. In this study, we developed a convenient gene disruption method in the yeast via Cre/loxP system. First, the simple gene disruption cassette [upstream homologous region (UP)-lox71-Sh ble-lox66-downstream homologous region (DW)] was constructed and transformed into the yeast to replace target gene. Second, the Sh ble gene of the cassette integrated in the chromosome was inserted with the auxiliary plasmid pPICZαA/cre/his4, resulting in an expanded cassette of UP-lox71-Sh ble-pPICZαA/cre/his4-lox66-DW. The auxiliary plasmid was generated via sequential insertion of cre and his4 genes into pPICZαA, and linearized with SmaI before its transformation. Finally, for deletion of the sequence between lox71 and lox66 sites in the expanded cassette, CRE protein responsible for Cre/loxP-mediated recombination was produced by methanol induction. Consequently, the corresponding sequence was eliminated permanently, only leaving a scar of lox72 site in the disrupted genes. This strategy was verified by disrupting two genes in the yeast. As the markers were recycled, it was also suitable for multiple gene disruption.

Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

BACKGROUND: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR). OBJECTIVE: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. METHOD: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. RESULTS: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. CONCLUSION: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.

Engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Determining cadmium bioavailability in sediment profiles using diffusive gradients in thin films.

Cadmium (Cd) uptake by plants or benthic organisms largely depends on its bioavailability in sediments, so it is necessary to understand Cd bioavailability for determining its ecological risks in riverine sediments. Pore water is easily disturbed during sample collection, indicating that there was a shortage of traditional methods for investigating Cd bioavailability. Here, sediment cores were collected from rivers, after which sequential extraction and diffusive gradients in thin films (DGT) method were employed to determine Cd potential bioavailability in the sediments and pore water. We found that Cd concentrations measured by DGT were lower than that in pore water profiles, and Cd distribution in various fractions changed remarkably. Pearson correlation analysis showed significant positive correlations between Cd concentrations measured by DGT and total Cd concentrations (r2 = 0.76), exchangeable and weak acid soluble fraction (r2 = 0.68), ferromanganese fraction (r2 = 0.72) and bound organic matter or oxidizable fraction (r2 = 0.54). However, the correlation was relatively low between Cd concentrations measured by DGT and that in pore water profiles (r2 = 0.26). These results demonstrated that DGT method could provide more accurate information of Cd bioavailability in sediment profiles than traditional methods.

Enhanced expression of heterologous proteins in yeast cells via the modification of N-glycosylation sites.

Yeasts are widely used for the production of heterologous proteins. Improving the expression of such proteins is a top priority for pharmaceutical and industrial applications. N-Glycosylation, a common form of protein modification in yeasts, facilitates proper protein folding and secretion. Accordingly, our previous study revealed that the attachment of additional N-glycans to recombinant elastase by introducing an N-glycosylation sequon at suitable locations could stimulate its expression. Interestingly, the sequon Asn-Xaa-Thr is N-glycosylated more efficiently than Asn-Xaa-Ser, so improving the N-glycosylation efficiency via the conversion of Ser to Thr in the sequon would enhance the efficiency of N-glycosylation and increase glycoprotein expression. Recently, the expression level of recombinant elastase was enhanced by this means in our lab. Actually, the modification of N-glycosylation sites can generally be achieved through site-directed mutagenesis; thus, the method described in this report represents a feasible means of improving heterologous protein expression in yeasts.

Enhanced expression of recombinant elastase in Pichia pastoris through the substitution of Thr for Ser in Asn-Xaa-Ser sequons.

N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.

Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide.

N-Glycosylation is a common form of protein post-translational modification in Pichia pastoris and greatly affects folding and secretion. The propeptide of the Pseudomonas aeruginosa elastase (PAE) is indispensable for proper folding and secretion of the enzyme. We have studied the effect of introducing N-glycosylation sites to the propeptide of the recombinant elastase (rPAE) on its expression levels in P. pastoris. Addition of N-glycosylation sites to the propeptide at N51 or N93 enhanced rPAE production levels by 104 or 57%, respectively, while addition at N11 or N127 led to a 25 or 50% decrease, respectively. The introduced N-glycosylation sites in the propeptide at these four sites exerted a null effect on the N-glycosylation degree of mature rPAE.

The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris.

The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70°C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.

Modification of recombinant elastase expressed in Pichia pastoris by introduction of N-glycosylation sites.

A novel N-glycosylation site was introduced into recombinant elastase (rPAE) at N36, N67, or N264 through the site-directed mutagenesis of I38T, A69T, or N266T, respectively. The A69T mutation completely inhibited the expression of rPAE. As expected, the I38T and N266T mutant proteins exhibited higher degrees of N-glycosylation compared with the wild type rPAE. The I38T mutant was more efficient in the hydrolysis of casein in aqueous medium and exhibited higher specific activity and k(cat) values and a lower K(m) value. In contrast, the N266T mutant and the wild type displayed similar values. Importantly, the I38T mutant achieved in higher rates and yields of peptide synthesis in 50% (v/v) dimethylsulfoxide, whereas the N266T mutant was similar to the wild type rPAE. Furthermore, the maximum yield of Z-Ala-Phe-NH2 synthesis catalyzed by the I38T mutant protein (87%) was higher than those achieved by the wild type (78%) and N266T mutant (78%) proteins. Neither the I38T nor the N266T mutation exerted significant effects on the rPAE solvent stability. In aqueous medium, the I38T mutation decreased the rPAE thermostability, and the N266T mutation slightly improved that. In conclusion, the I38T mutation improved the potential of rPAE in industrial applications.