Dataset Information

Engineering Heterologous Production of Salicylate Glucoside and Glycosylated Variants.

ABSTRACT: Salicylate 2-O-?-D-glucoside (SAG) is a plant-derived natural product with potential utility as both an anti-inflammatory and as a plant protectant compound. Heterologous biosynthesis of SAG has been established in Escherichia coli through metabolic engineering of the shikimate pathways and introduction of a heterologous biosynthetic step to allow a more directed route to the salicylate precursor. The final SAG compound resulted from the separate introduction of an Arabidopsis thaliana glucosyltransferase enzyme. In this study, a range of heterologous engineering parameters were varied (including biosynthetic pathway construction, expression plasmid, and E. coli strain) for the improvement of SAG specific production in conjunction with a system demonstrating improved plasmid stability. In addition, the glucoside moiety of SAG was systematically varied through the introduction of the heterologous oliose and olivose deoxysugar pathways. Production of analogs was observed for each newly constructed pathway, demonstrating biosynthetic diversification potential; however, production titers were reduced relative to the original SAG compound.

SUBMITTER: Qi R

PROVIDER: S-EPMC6158457 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Engineering Heterologous Production of Salicylate Glucoside and Glycosylated Variants.

Qi Ruiquan R Pfeifer Blaine A BA Zhang Guojian G

Frontiers in microbiology 20180920

Salicylate 2-O-β-D-glucoside (SAG) is a plant-derived natural product with potential utility as both an anti-inflammatory and as a plant protectant compound. Heterologous biosynthesis of SAG has been established in <i>Escherichia coli</i> through metabolic engineering of the shikimate pathways and introduction of a heterologous biosynthetic step to allow a more directed route to the salicylate precursor. The final SAG compound resulted from the separate introduction of an <i>Arabidopsis thaliana ...[more]

PMID: 30294315

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Project description:Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Project description:BACKGROUND: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. RESULTS: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man?GlcNAc? or Man?GlcNAc?. First, we inactivated the yeast-specific Golgi ?-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man?GlcNAc? glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the ?-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained ?-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man?GlcNAc?. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. CONCLUSIONS: We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man?GlcNAc? or Man?GlcNAc? N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.

Dataset Information

Engineering Heterologous Production of Salicylate Glucoside and Glycosylated Variants.

Publications

Engineering Heterologous Production of Salicylate Glucoside and Glycosylated Variants.

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