Engineering Corynebacterium glutamicum for high-titer biosynthesis of hyaluronic acid

Cheng, Fangyu; Yu, Huimin; Stephanopoulos, Gregory

doi:10.1016/j.ymben.2019.07.003

Cited by 86 publications

(54 citation statements)

References 58 publications

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“…1b). Protein alignment showed seHasA 21 and sseHasA 22,29,31 share the same protein sequence. In comparison, there is 72% sequence identity between seHasA and spHasA and 71% sequence identity between suHasA and spHasA ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to overexpressing UgdA and GlmS for precursor generation (Fig. 1a), deletion of the competing pathway to increase the supply of UDP-glucuronate and UDP-GlcNAc is a commonly used strategy 22,29 . In C. glutamicum, there is less availability of UDP-GlcA relative to UDP-GlcNAc, given that overexpression of UgdA improved HA yield by 2-fold (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The development of metabolic engineering allows the engineering of non-pathogenic Escherichia coli strains for heterologous HA production 10,11 . To further eliminate potential safety concerns, many generally recognized as safe (GRAS) strains, such as Bacillus subtilis [12][13][14][15][16] , Lactococcus lactis [17][18][19][20] , Corynebacterium glutamicum 21,22 , and Pichia pastoris 23 , have been engineered as alternative producers of HA. Additionally, cell-free systems have been exploited to produce HA at specific molecular weight (MW) 24,25 .…”

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confidence: 99%

“…Additionally, deletion of ldh, encoding lactate dehydrogenase, increased HA production to 21.6 g L −1 in fed-batch culture 29 . A follow-up study enhanced the HA yield to 28.7 g L −1 by attenuating the glycolysis pathway, pentose phosphate pathway and the dehydrogenation of pyruvate 22 .…”

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confidence: 99%

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

Wang

Huang

et al. 2020

Nat Commun

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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 fedbatch 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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

Wang

Huang

et al. 2020

Nat Commun

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“…Unlike streptococcus , C. glutamicum is a Generally-Recognized- as -Safe (GRAS) strain widely used in the food industry [ 14 ]. By introducing the HAS genes and optimizing the metabolic pathways, the high titer production of HA (28.7 g/L, 210kD Mw) was achieved in a 5 L fermentor with the engineered C. glutamicum ATCC13032 [ 15 ]. Further by introducing into the hyaluronidase, 74 g/L small 54kD Mw HA was also obtained [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancing single-cell hyaluronic acid biosynthesis by microbial morphology engineering

Zheng

Cheng

Zheng

et al. 2020

Synthetic and Systems Biotechnology

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Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture. Hyaluronic acid (HA), a valuable glycosaminoglycan polymerized by HA synthase (HAS), a membrane protein, is particularly selected as the model product to improve its single-cell HA-producing capacity via morphology engineering. DivIVA and FtsZ, the cell-elongation and cell division related protein, respectively, were both down/up dual regulated in C. glutamicum via weak promoter substitution or plasmid overexpression. Different from the natural short-rod shape, varied morphologies of engineered cells, i.e. small-ellipsoid-like (DivIVA-reduced), bulb-like (DivIVA-enhanced), long-rod (FtsZ-reduced) and dumbbell-like (FtsZ-enhanced), were observed. Applying these morphology-changed cells as hosts for HA production, the reduced expression of both DivIVA and FtsZ seriously inhibited normal cell growth; meanwhile, overexpression of DivIVA didn't show morphology changes, but overexpression of FtsZ surprisingly change the cell-shape into long and thick rod with remarkably enlarged single-cell surface area (more than 5.2-fold-increase). And finally, the single-cell HA-producing capacity of the FtsZ-overexpressed C. glutamicum was immensely improved by 13.5-folds. Flow cytometry analyses verified that the single-cell HAS amount on membrane was enhanced by 2.1 folds. This work is pretty valuable for high titer synthesis of diverse metabolic products with microbial cell factory.

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Metabolic Engineering of Corynebacterium glutamicum

Becker

Wittmann

2021

Metabolic Engineering

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Engineering Corynebacterium glutamicum for high-titer biosynthesis of hyaluronic acid

Cited by 86 publications

References 58 publications

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

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

Enhancing single-cell hyaluronic acid biosynthesis by microbial morphology engineering

Metabolic Engineering of Corynebacterium glutamicum

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