Dataset Information

High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide.

ABSTRACT: An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL) in yeast culture supernatant reached 226.4 ?g/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K? ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T5015 of 57?58 °C and T5030 of 53?54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by P. pastoris attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using P. pastoris system.

SUBMITTER: Li H

PROVIDER: S-EPMC5983289 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide.

Li Haifeng H Wang Shuling S Zhang Yunyi Y Chen Liehuan L

Marine drugs 20180511 5

An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL) in yeast culture supernatant reached 226.4 μg/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. T ...[more]

PMID: 29751659

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Project description:BackgroundThe conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming.ResultsThe gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K⁺, Li⁺, Ni²⁺ and Sr²⁺ showed little or no inhibitory effect on PEL168P activity, and Ca²⁺ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization.ConclusionsPectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming.

Project description:The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Project description:Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism.The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking.This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Dataset Information

High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide.

Publications

High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide.

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