Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis

Jin, Peng; Zhang, Linpei; Yuan, Panhong; Kang, Zhen; Du, Guocheng; Chen, Jian

doi:10.1016/j.carbpol.2015.12.065

Cited by 88 publications

(63 citation statements)

References 57 publications

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“…As shown in Figure b, consistent with our previous study (Jin, Zhang, et al, ), up‐regulation of tuaD (encoding UDP‐glucose dehydrogenase) significantly increased chondroitin production from 1.63 ± 0.09 to 2.36 ± 0.12 g/L, confirming UDP‐glucose dehydrogenase is a rate‐limiting enzyme towards the biosynthesis of UDP‐GlcUA. In contrast, further co‐overexpression of gtaB (encoding pyrophosphorylase) diminished chondroitin accumulation (2.15 ± 0.05 g/L).…”

Section: Resultssupporting

confidence: 91%

“…E. coli JM109 was used as host for gene cloning. The genes tuaD , glmU , gtaB , glmM , glmS , and kfoA were amplified from the genomic DNA of B. subtilis E168C (Jin, Zhang, et al, ) by polymerase chain reaction (PCR) using PrimeSTAR HS (Premix) (Takara Bio Inc., Shiga, Japan) with the primers in Supplementary Table S2. First, the fragments Kpn I‐ tuaD ‐ Sac I‐ Xho I and Xba I‐ glmU ‐ Nhe I‐ Xho I were ligated into pP43NMK with Kpn I/ Xho I and Xba I/ Xho I, generating plasmids pP43‐D, pP43‐U, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Nevertheless, E. coli K4 is a pathogenic strain, which causes urinary tract infection (Nash et al, ). Thus, the chondroitin biosynthesis pathway has been rebuilt and optimized in other strains such as E. coli BL21(DE3) (He et al, ), and Bacillus subtilis 168 (Jin, Zhang, et al, ) and the hyaluronan producing strain Streptococcus zooepidemicus (Marcellin, Chen, & Nielsen, ). Additionally, a microbiological–chemical strategy was established for synthesis of CS by removing the β‐fructose residue at the O‐3 position of the GlcA units and chemical addition of the sulfation group at positions 4 and 6 of the GalNAc residues (Fujikawa et al, ).…”

Section: Introductionmentioning

confidence: 99%

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A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

Zhou

Huang

et al. 2018

Biotech & Bioengineering

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Chondroitin sulfate has been widely used in both medical and clinical applications. Commercial chondroitin sulfate has been mainly acquired from animal tissue extraction. Here we report a new two-step biological strategy for producing chondroitin sulfate A and chondroitin sulfate C. First, the chondroitin biosynthesis pathway in a recombinant Bacillus subtilis strain using sucrose as carbon source was systematically optimized and the titer of chondroitin was significantly enhanced to 7.15 g/L. Then, specific sulfation transformation systems were successfully constructed and optimized by combining the purified aryl sulfotransferase IV (ASST IV), chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST). Chondroitin sulfate A and C were enzymatically transformed from chondroitin at conversion rates of 98% and 96%, respectively. The present biological strategy has great potential to be scaled up for biosynthesis of chondroitin sulfate A and C from cheap carbon sources.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

Zhou

Huang

et al. 2018

Biotech & Bioengineering

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“…In addition, its precursor chondroitin, was more recently shown to reduce the inflammatory response in IL‐1β‐treated chondrocytes and enhance their proliferation and phenotype preservation (Stellavato et al, ). In this framework, several efforts have been made aimed to improve polymer production by genetically engineering the natural producer E. coli K4 or other appealing host strains (Cimini, De Rosa, Carlino, Ruggiero, & Schiraldi, ; Cimini, De Rosa, et al, ; Cimini et al, ; He et al, ; Jin et al, ; Wu et al, ) to obtain valid alternatives to currently used animal‐derived sources. The genes responsible for capsular polysaccharide (CPS) biosynthesis and transport in E. coli K4 are clustered and organized in three regions; regions 1 and 3 are common to all group II E. coli strains, and encode genes involved in the export and assembly of CPS on the cell surface (Rigg, Barrett, & Roberts, ).…”

Section: Introductionmentioning

confidence: 99%

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

Cimini

Russo

D’ambrosio

et al. 2018

Biotech & Bioengineering

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Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP-sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP-glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild-type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP-sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state-of-the-art quantitative proteomic approaches, as well as genetic and physiological information.

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“…K4 and K5 polysaccharides are consolidated precursors of chondroitin and heparin; however, CS is still obtained from limited and potentially harmful, animal sources such as chicken keel, shark fins, and swine or bovine trachea. Several efforts have been made to use E. coli K4, or its biosynthetic machinery in a different background, to generate cell factories for the production of chondroitin, and encouraging results were obtained (Cimini et al 2013, 2015; He et al 2015; Jin et al 2016). The reason lies in the fact that the capsular polysaccharide of this uropathogenic strain, composed of alternating residues of N -acetylgalactosamine (GalNAc) and glucuronic acid (GlcA), has a structure that closely resembles the backbone of CS.…”

Section: Introductionmentioning

confidence: 99%

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

et al. 2017

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Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitin-like polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0364-7) contains supplementary material, which is available to authorized users.

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Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis

Cited by 88 publications

References 57 publications

A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

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