Compact multi-enzyme pathways in P. pastoris (original) (raw)
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Functional Representation of Enzymes by Specific Peptides
PLoS Computational Biology, 2007
Predicting the function of a protein from its sequence is a long-standing goal of bioinformatic research. While sequence similarity is the most popular tool used for this purpose, sequence motifs may also subserve this goal. Here we develop a motif-based method consisting of applying an unsupervised motif extraction algorithm (MEX) to all enzyme sequences, and filtering the results by the four-level classification hierarchy of the Enzyme Commission (EC). The resulting motifs serve as specific peptides (SPs), appearing on single branches of the EC. In contrast to previous motif-based methods, the new method does not require any preprocessing by multiple sequence alignment, nor does it rely on over-representation of motifs within EC branches. The SPs obtained comprise on average 8.4 6 4.5 amino acids, and specify the functions of 93% of all enzymes, which is much higher than the coverage of 63% provided by ProSite motifs. The SP classification thus compares favorably with previous function annotation methods and successfully demonstrates an added value in extreme cases where sequence similarity fails. Interestingly, SPs cover most of the annotated active and binding site amino acids, and occur in active-site neighboring 3-D pockets in a highly statistically significant manner. The latter are assumed to have strong biological relevance to the activity of the enzyme. Further filtering of SPs by biological functional annotations results in reduced small subsets of SPs that possess very large enzyme coverage. Overall, SPs both form a very useful tool for enzyme functional classification and bear responsibility for the catalytic biological function carried out by enzymes. Citation: Kunik V, Meroz Y, Solan Z, Sandbank B, Weingart U, et al. (2007) Functional representation of enzymes by specific peptides. PLoS Comput Biol 3(8): e167.
Biological roles of specific peptides in enzymes
Proteins: Structure, Function, and Bioinformatics, 2008
Specific Peptides (SPs) have been shown [22] to specify the functions of 93% of the enzymes on which they occur. In this work we will focus on the biological importance and possible roles of SPs in the realisation of enzymatic functions. SPs are shown to provide correct functional classification in problems where conventional methods, based on sequence or structure similarity, fail. These cases include enzymatic functions that diverged or converged with evolution. I am greatly indebted to my advisor, Prof. David Horn, who showed me the fun in delving into things I do not know and do not understand. As Dante Alighieri put it in La Divina Commedia (Inferno, Canto XI); "O sol che sani ogni vista turbata, tu mi contenti sì quando tu solvi, che, non men che saver, dubbiar m'aggrata." "O Sun that healest all dim sight, thou so dost charm me in resolving of my doubt, to be perplexed is pleasant as to know." I would also like to thank my lab-mates-Assaf, Liat, Roy, Uri, Vered, Yaron and Zach-who made me smile every day, infallibly. Last, but certainly not least, I thank my parents, sisters and grandmother for their constant love and support. And then there's Ely, who always believed in me more than I did myself.
Gene, 2017
The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.
Molecular BioSystems, 2008
Non-ribosomal peptide products often contain modified building blocks or postassembly line alterations of their peptide scaffolds with some of them being crucial for biological activity. These reactions such as halogenation, hydroxylation or glycosylation are mostly catalyzed by individual enzymes associated with the respective biosynthesis cluster. The versatile nature of these chemical modifications gives rise to a high degree of structural and functional diversity. Recent progress in this area enhances our insight about the mechanisms of these enzymes. Biotechnological applications might include the synthesis of novel, non-ribosomal peptide products or modified amino acid building blocks for pharmaceutical research.
The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates
Journal of Biological Chemistry, 1996
Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.
2021
Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strate...
A High-throughput Approach to NRPS Domain Substitution for Generation of Novel Peptides
Many small, medically significant peptides are produced by non-ribosomal peptide synthetases (NRPSs). NRPSs are large modular enzymes that function as an assembly-line with each module adding one amino acid subunit to the final product. Each module is comprised of domains which perform the functions of substrate specification, peptide bond formation; and substrate loading and transfer. Novel peptides can be produced by modifying the NRPS template to enable a different amino acid to be incorporated into the peptide product. However, previous efforts at engineering these enzymes have been inefficient and not scalable, frequently resulting in non-functional proteins. This study focussed on development of a powerful high-throughput method for the targeted mass modification of NRPS templates. A pyoverdine synthesising NRPS from Pseudomonas aeruginosa PAO1 was used as a model system. Pyoverdine is a fluorescent green siderophore essential for growth on iron-restricted media thus it is ...
BMC Biotechnology, 2010
Background: Penicillin G acylase of Escherichia coli (PGA Ec ) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGA Ec in this host and studied yeast production capacity and enzyme authenticity.
A high-throughput expression screening platform to optimize the production of antimicrobial peptides
Microbial Cell Factories
Background: Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. Results: The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His 6 tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression. Conclusions: The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression.