18 research outputs found
Exploring alternative expression systems for expression of heterologous proteins and metabolic pathways
No full textAugust 2016School of ScienceOur next goal was to use chromosomal integration for metabolic engineering. Multiple studies, from our laboratory and others, have found that modulatin of gene expression is key to pathway optimization. Generally, this has been achieved using promoters of varying strengths on plasmids of variable copy number. However, chromosomal integration offers advantages for metabolic engineering that could resilt in increased production of high-value chemical products. Here, we have shown, usnig a model five-gene pathway for the production of the purple pigment violacein from tryptophan, again that the location of the integration matters. We found that of the four genomic locations used, only one showed any level of violacein production, and it was surprisingly not the same location that resulted in increased levels of mCherry expression. Interestingly, when the one-gene TAL pathway, which converts phenylalanine to trans-cinnamic acid, was integrated into the same loci, very little difference in production was observed between the locations. These results indicate that optimization of production may not only be location dependent, but it may also depend on the genes being expressed.Next, we attempted to find an improved expression system for the heparin biosynthetic enzymes. To this end, two species of Bacillus and the yeast Pichia pastoris were tested for their ability to secrete the heparin biosynthetic enzymes. While intracellular expression of enzymatically active 3-OST-1 was achieved in the Bacillus species, no level of secretion was measured using a small library of signal peptides in Bacillus megaterium. Successful secretion of all of the sulfotransferases required for the chemoenzymatic synthesis of heparin was achieved in P. pastoris. Here, we hae shown that when expressed as glycoproteins in P. pastoris, these enzymes show favorable sulfotransferase activity compared to their E. coli-expressed counterparts. These results indicate that the post-translational modification of protein glycosylation plays an important role in the activity of the heparin biosynthetic enzymes and that the yeast P. pastoris may be a good host for the expression and secretion of the heparin biosynthetic enzymes.Heparin is the most widely used anticoagulant drug in the world. Current production of heparin from porchine intestinal tissue led to a contamination of the heparin supply in 2008 that resulted in over 100 deaths. This, coupled with the demand heparin puts on the worldwide pig population, has led to the need of non-animal sourced heparin. A chemeoenzymatic synthetic scheme, developed in our laboratory, has previously been used to successfully produce anticoagulant heparin. However, the enzymes for this synthesis are currently expressed and purified from Escherchia coli utilizing plasmid-based expression vectors. This Gram-negative organism is widely used for heterologous protein expression because of its fast growth and the vast molecular toolkit avilable. The work here aimed to alleviate problems with plasmid-based expression in E. coli.Heterologous protein expression has long been carried out using plasmids as expression vectors in E. coli. Problems with plasmid-based expression, primarily the necessity for selection using antibiotics that can be expensive and deleterious to the environment, led to the desire to integrate the genes for the chemoenzymatic synthesis of heparin on the genomic chromosome of E. coli. While the expression of these enzymes was unsuccessful from the genome, it became apparent that chromosomal integration had other uses. Here, we have used model reporter proteins and pathways to show that there is much to learn about chromosomal integration. By integrating the gene encoding the fluorescent reporter protein mCherry into various loci throughout the genome of E. coli, we found that expression level was location-dependent. Further, we found that integration of a single copy of the gene into one particular locus resulted in over 5-fold higher mCherry expression than a high-copy plasmid expressing the same gene. We had hypothesized that to achieve the same level of expression found in plasmid-based systems, we would need to increase the copy number of the gene. Surprisingly, we found that when we increased the copy number of the gene in a single genomic locus, there was actually a decrease in mCherry expression.Ph
Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules
No full textFEMS Microbiology Reviews, 38, 660–697Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described, providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment.National Heart, Lung, and Blood Institutehttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1111/1574-6976.1205
Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis
No full textBiochemistry, 57, 1190−1200Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC’s tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acs.biochem.7b0118
CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli
No full textACS Synthetic Biology, 4, 987–1000Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions.National Science Foundationhttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/acssynbio.5b0001
Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris
No full textApplied Microbiology and Biotechnology, 101, 2843–2851Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.National Science Foundationhttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1007/s00253-016-8047-
Expression of low endotoxin 3-O-sulfotransferase in Bacillus subtilis
No full textApplied Biochemistry and Biotechnology, 171, 954–962Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.National Institutes of Healthhttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1007/s12010-013-0415-
Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli
No full textChromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway
Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices
No full textAnaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery
Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli
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