Yuhong Zhang

High-yield production of a low-temperature-active polygalacturonase for papaya juice clarification

A novel endo-polygalacturonase (endo-PG I) from Achaetomium sp. Xz8 was identified, overexpressed in Pichia pastoris, and characterized in this report. Recombinant endo-PG I is distinguished from other enzyme counterparts by its high activity towards polygalacturonic acid (49,934 U/ml) and high yield in the 15-l fermentor (2.13 g/l). It exhibits optimal activity at 45 °C and remained active over a broad temperature range of 0-80 °C. Distinct from most fungal polygalacturonases that have acidic pH optima, endo-PG I is optimally active at pH 6, similar to the pH of fresh papaya juice (5.7). Endo-PG I alone reduced the viscosity of papaya juice by 17.6%, and increased its transmittance by 59.1%. When combined with a commercial pectin methylesterase, it showed much higher efficiency with a synergy degree of more than 1.25. All these favourable enzymatic properties make endo-PG I attractive for potential applications in the juice industry.

RNA-Seq-Based Analysis of Cold Shock Response in Thermoanaerobacter tengcongensis, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene

Background Although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC), can survive from 50° to 80°C, but has poor natural competence under cold shock at 50°C. We therefore examined cold shock responses and their effect on natural competence in this bacterium. Results The transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature. Conclusion Our study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic and molecular mechanisms associated with cold shock and acclimation at low temperature.

Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris

The rhamnose utilization pathway in Pichia pastoris has not been clarified although this strain can grow well on rhamnose as a sole carbon source. In this study, four genes, PAS_chr4_0338, PAS_chr4_0339, PAS_chr4_0340, and PAS_chr4_0341, were, for the first time, predicted to be involved in rhamnose metabolism along with the previously identified gene PAS_chr1_4-0075. Moreover, expression of these genes, especially PAS_chr4_0341 and PAS_chr1_4-0075 designated as LRA4 and LRA3, was confirmed to significantly increase and clearly decrease in the presences of rhamnose and glucose, respectively. LRA4 encoding a putative L-2-keto-3-deoxyrhamnonate aldolase, was further confirmed via gene disruption and gene complementation to participate in rhamnose metabolism. Using β-galactosidase and green fluorescent protein as reporters, the promoters of LRA4 and LRA3 performed well in driving efficient production of heterologous proteins. By using food grade rhamnose instead of the toxic compound methanol as the inducer, the two promoters would be excellent candidates for driving the production of food-grade and therapeutically important recombinant proteins.

Production and secretion of Lactobacillus crispatus β-galactosidase in Pichia pastoris

Lactobacillus β-galactosidases are mostly heterodimeric proteins, which are encoded by the two overlapping genes, lacL and lacM, and produced in recombinant prokaryotic systems for higher yield. This is the first report on the expression of a heterodimeric β-galactosidase from Lactobacillus crispatus B470 in Pichia pastoris. The overlapping consecutive genes, lacL and lacM, that shared 17 nucleotides were cloned from the genomic DNA of L. crispatus. A recombinant plasmid harboring both expression cassettes of lacL and lacM was constructed and transformed into P. pastoris GS115 competent cells. Two recombinant P. pastoris strains (GSLac01 and GSLac02) showed the highest β-galactosidase activities of 24.5 and 31.0 U/ml in the culture supernatants, respectively. The recombinant β-galactosidase (LcLacLM) from GSLac02 was purified to electrphoretic homogeneity by ion-exchange chromatography and molecular sieve chromatography. Similar to most Lactobacillus β-galactosidases that operate at moderately thermophilic and weak acid to neutral conditions, LcLacLM showed optimal activity at 50°C and pH 5.5-6.5. Its the first report on functional and secretory expression of LacLM-type β-galactosidase in eukaryotic system. This strategy might be applied to the expression of other overlapping genes.

Overexpression of a Fungal β-Mannanase from Bispora sp. MEY-1 in Maize Seeds and Enzyme Characterization

Background Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production. Methodology/Principal Findings The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C. Conclusion/Significance This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

Background Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed. Methodology/Principal Findings Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract. Conclusion/Significance Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50°C) and lower stability over alkaline pH range, but better thermal stability at 60°C to 70°C and resistance to feed pelleting inactivation (80°C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

Efficient Expression of an Acidic Endo-polygalacturonase from Aspergillus niger and Its Application in Juice Production

An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was 1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris GS115 for heterologous expression. The polygalacturonase showed a maximum activity level of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were 4.5 and 40 °C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the volume of pear juice by 41.8%, and improved its light transmittance 3-fold.

Presep: predicting the propensity of a protein being secreted into the supernatant when expressed in Pichia pastoris.

Pichia pastoris is commonly used for the production of recombinant proteins due to its preferential secretion of recombinant proteins, resulting in lower production costs and increased yields of target proteins. However, not all recombinant proteins can be successfully secreted in P. pastoris. A computational method that predicts the likelihood of a protein being secreted into the supernatant would be of considerable value; however, to the best of our knowledge, no such tool has yet been developed. We present a machine-learning approach called Presep to assess the likelihood of a recombinant protein being secreted by P. pastoris based on its pseudo amino acid composition (PseAA). Using a 20-fold cross validation, Presep demonstrated a high degree of accuracy, with Matthews correlation coefficient (MCC) and overall accuracy (Q2) scores of 0.78 and 95%, respectively. Computational results were validated experimentally, with six β-galactosidase genes expressed in P. pastoris strain GS115 to verify Presep model predictions. A strong correlation (R2 = 0.967) was observed between Presep prediction secretion propensity and the experimental secretion percentage. Together, these results demonstrate the ability of the Presep model for predicting the secretion propensity of P. pastoris for a given protein. This model may serve as a valuable tool for determining the utility of P. pastoris as a host organism prior to initiating biological experiments. The Presep prediction tool can be freely downloaded at http://www.mobioinfor.cn/Presep.