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Fulltext Comparative Genomics: Insights on the Pathogenicity and Lifestyle of Rhizoctonia solani

Mat Razali N, Hisham SN, Kumar IS, Shukla RN, Lee M, Abu Bakar MF, et al.

Int J Mol Sci, 2021 Feb 22;22(4).
PMID: 33671736 DOI: 10.3390/ijms22042183

Proper management of agricultural disease is important to ensure sustainable food security. Staple food crops like rice, wheat, cereals, and other cash crops hold great export value for countries. Ensuring proper supply is critical; hence any biotic or abiotic factors contributing to the shortfall in yield of these crops should be alleviated. Rhizoctonia solani is a major biotic factor that results in yield losses in many agriculturally important crops. This paper focuses on genome informatics of our Malaysian Draft R. solani AG1-IA, and the comparative genomics (inter- and intra- AG) with four AGs including China AG1-IA (AG1-IA_KB317705.1), AG1-IB, AG3, and AG8. The genomic content of repeat elements, transposable elements (TEs), syntenic genomic blocks, functions of protein-coding genes as well as core orthologous genic information that underlies R. solani's pathogenicity strategy were investigated. Our analyses show that all studied AGs have low content and varying profiles of TEs. All AGs were dominant for Class I TE, much like other basidiomycete pathogens. All AGs demonstrate dominance in Glycoside Hydrolase protein-coding gene assignments suggesting its importance in infiltration and infection of host. Our profiling also provides a basis for further investigation on lack of correlation observed between number of pathogenicity and enzyme-related genes with host range. Despite being grouped within the same AG with China AG1-IA, our Draft AG1-IA exhibits differences in terms of protein-coding gene proportions and classifications. This implies that strains from similar AG do not necessarily have to retain similar proportions and classification of TE but must have the necessary arsenal to enable successful infiltration and colonization of host. In a larger perspective, all the studied AGs essentially share core genes that are generally involved in adhesion, penetration, and host colonization. However, the different infiltration strategies will depend on the level of host resilience where this is clearly exhibited by the gene sets encoded for the process of infiltration, infection, and protection from host.

Matched MeSH terms: Protein Sorting Signals/genetics
Fulltext Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis

Koko I, Song AA, Masarudin MJ, Abdul Rahim R

BMC Biotechnol, 2019 11 27;19(1):82.
PMID: 31775775 DOI: 10.1186/s12896-019-0575-x

BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system.
RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.
CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

Matched MeSH terms: Protein Sorting Signals/genetics
Fulltext Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli: Is there an optimal signal peptide design?

Low KO, Jonet MA, Ismail NF, Illias RM

Bioengineered, 2012 Nov-Dec;3(6):334-8.
PMID: 22892592 DOI: 10.4161/bioe.21454

Recombinant protein fused to an N-terminal signal peptide can be translocated to the periplasm and, eventually, to the extracellular medium of Escherichia coli under specific conditions. In this communication, we described the use and optimization of a heterologous signal peptide (G1 signal peptide) from a Bacillus sp for improved recombinant protein secretion and cell viability in E. coli. Significant advantages in maintaining high cell viability and high specificity of target protein secretion were achieved by using G1 signal peptide compared to the well-known PelB signal peptide. Signal peptide sequence analysis and site-directed mutagenesis of G1 signal peptide demonstrated that an 'MKK' sequence in n-region and the presence of a helix-breaking residue at the centre of h-region are important elements for the design of an optimal signal peptide.

Matched MeSH terms: Protein Sorting Signals/genetics*
A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli

Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al.

Biotechnol Lett, 2011 May;33(5):999-1005.
PMID: 21234789 DOI: 10.1007/s10529-011-0517-8

L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.

Matched MeSH terms: Protein Sorting Signals/genetics*
Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase

Ismail A, Illias RM

J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
PMID: 28921081 DOI: 10.1007/s10295-017-1980-6

The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.

Matched MeSH terms: Protein Sorting Signals/genetics*
Secretion of recombinant xylanase in Lactococcus lactis using signal peptides Usp45 and Spk1

Roslan AM, Mustafa Kamil A, Chandran C, Song AA, Yusoff K, Abdul Rahim R

Biotechnol Lett, 2020 Sep;42(9):1727-1733.
PMID: 32335791 DOI: 10.1007/s10529-020-02894-1

OBJECTIVE: The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed.
RESULTS: Xylanase was successfully expressed in Lactococcus lactis. Recombinant xylanase fused to either signal peptide Usp45 or Spk1 showed halo zone on Remazol Brilliant Blue-Xylan plates. This indicated that the xylanase was successfully secreted from the cell. The culture supernatants of strains secreting the xylanase with help of the Spk1 and Usp45 signal peptides contained 49.7 U/ml and 34.4 U/ml of xylanase activity, respectively.
CONCLUSION: Although Usp45 is the most commonly used signal peptide when secreting heterologous proteins in Lactococcus lactis, this study shows that Spk1 isolated from Pediococcus pentosaceus was superior to Usp45 in regard to xylanase protein secretion.

Matched MeSH terms: Protein Sorting Signals/genetics*
Fulltext Permanent neonatal diabetes due to a novel insulin signal peptide mutation

Hussain S, Mohd Ali J, Jalaludin MY, Harun F

Pediatr Diabetes, 2013 Jun;14(4):299-303.
PMID: 23350652 DOI: 10.1111/pedi.12011

We report a rare case of permanent neonatal diabetes (PND) due to insulin (INS) gene mutation in a 51-month-old girl who presented with hyperglycemia in the neonatal period. Mutational analysis of KCNJ11 and INS was performed and this detected a novel heterozygous c.38T>G (p.Leu13Arg) INS de novo mutation. The non-conservative change substitutes the highly conserved L(13) residue within the hydrophobic core region of the preproinsulin signal peptide. Given the frequent tendency of heterozygous INS mutations to exhibit dominant negative disease pathogenesis, it is likely that the mutant preproinsulin perturbed the non-mutant counterpart progression and processing within the β-cells, and this resulted to a permanent form of congenital diabetes.

Matched MeSH terms: Protein Sorting Signals/genetics
Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli

Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RN, Illias RM

J Ind Microbiol Biotechnol, 2008 Dec;35(12):1705-14.
PMID: 18726621 DOI: 10.1007/s10295-008-0462-2

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.

Matched MeSH terms: Protein Sorting Signals/genetics
Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y

Masomian M, Jasni AS, Rahman RNZRA, Salleh AB, Basri M

J Biotechnol, 2017 Dec 20;264:51-62.
PMID: 29107669 DOI: 10.1016/j.jbiotec.2017.10.014

A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.

Matched MeSH terms: Protein Sorting Signals/genetics*