Dataset Information

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

ABSTRACT: Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2?g?L-1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1?g?L-1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7305114 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

Wang Yang Y Hu Litao L Huang Hao H Wang Hao H Zhang Tianmeng T Chen Jian J Du Guocheng G Kang Zhen Z

Nature communications 20200619 1

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways an ...[more]

PMID: 32561727

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Project description:BackgroundExtremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing.ResultsHere, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L-1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purification.ConclusionsC. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.

Project description:In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly l-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. l-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc IMPORTANCE: Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.

Dataset Information

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

Publications

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

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