Production of Hyaluronic Acid by Streptococcus

Ogrodowski, Christiane Saraiva; Hokka, Carlos O.; Santana, Maria Helena Andrade

doi:10.1007/978-1-59259-991-2_63

Cited by 4 publications

(6 citation statements)

References 4 publications

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“…Fermentation broth samples were incubated first with an equal volume of 0.1% w/v sodiumdodecyl-sulfate (SDS) at room temperature for 10 min to free the capsular HA (Chong and Nielsen, 2003). Subsequently, the HA product was precipitated out from the medium samples with 2 volumes of ethanol (Ogrodowski et al, 2005) incubating at 48C for 1 h. The precipitate was collected by centrifugation (2,000g for 20 min at room temperature) and resuspended in 1 volume of deioned water for 10 min. Then the re-dissolved samples were applied to the modified CTAB assay as follows: add 0.15 mL sample into 0.35 mL acetate buffer, mix then add 1.0 mL CTAB reagent; mix and incubate at room temperature for 5 min; measure OD 400 and calculate the HA titer by 20Â the value calculated from the standard curve, which was prepared by assaying the correlation of the turbidity OD 400 to different concentration of HA diluted by a concentrated 0.5 g/L standard with MW of 6.8 Â 10 5 (Lifecore Biomedical, Inc., Chaska, MN).…”

Section: Ha Titer Measurement By a Modified Ctab Methodsmentioning

confidence: 99%

“…Its importance stems from its structural, rheological, physiological, and biological properties, leading to a wide range of applications in the health, cosmetic and clinical fields (Goa and Benfield, 1994;Lauren, 1998). Microbial production of HA using the group C or group A Streptococcus has been pursued as alternative to chemical extraction from chicken comb (DeAngelis et al, 1993;Kim et al, 1996;Kumari and Weigel, 1997;Kakizaki et al, 2002;Ogrodowski et al, 2005). Recently, new methods using novel recombinant production strains that utilize inexpensive media and avoid pathogenicity have been developed as substitute of Streptococcus fermentation.…”

Section: Introductionmentioning

confidence: 98%

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A high‐throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

Tyo

Alper

et al. 2008

Biotech & Bioengineering

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Hyaluronic acid (HA) is an important biomaterial with functional medical and cosmetic applications. As its synthesis has been recently reported in recombinant bacteria, it is of interest to develop a high throughput screening method for the rapid isolation of HA accumulating strains transformed by combinatorial libraries. Here we report a novel two-step screening strategy to select for better HA-producing recombinant Escherichia coli strains transformed by mutation libraries of rpoD and rpoS, coding for the sigma(D) and sigma(S) factors of the RNA polymerase, respectively. The first screen, based on translucent colony morphology identification, was used to qualitatively distinguish HA-producing strains on agar plates from non-HA producing strains that exhibit dense colony morphology. The second screen was based on the photometric measurement of an alcian blue staining solution that precipitates with HA, creating an inverse relationship between HA concentration and alcian blue absorbance. The color attenuation fitted a second-order polynomial between HA concentration and OD(540) absorbance. Using the alcian blue absorbance quantification, 74 translucent colonies from the HA-rpoD library and 78 translucent colonies from the HA-rpoS library were isolated and cultured for optimal strain selection. Three representative superior recombinants with high, medium and low increase of HA accumulation, respectively, were identified by the screen from the HA-rpoD and HA-rpoS mutant library. Further flask culture confirmed that results of the library screen were reliable and the superior recombinant D72 highly accumulated HA of 561.4 mg/L with a productivity of approximately 265 mg HA/g dry cell. Sequencing results showed that the mutant rpoD gene in D72 is in a truncated protein that lacks the conserved regions 3 and 4 of the sigma(D). Generally, this two-step high throughput screen presents a promising strategy for selecting superior HA-producing strains from large scale mutation libraries.

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Section: Ha Titer Measurement By a Modified Ctab Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

A high‐throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

Tyo

Alper

et al. 2008

Biotech & Bioengineering

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“…The original method to produce HA was chemical extraction from chicken combs. It was plagued by the low productivity and requirement for complex purification steps [6]. A microbial fermentation method was developed in Japan for accumulation of HA via Streptococcus in the 1980s [6][7][8][9], and a novel bio-route by fermentation of the engineered strains was proposed in recent years [10][11][12][13][14][15][16].…”

mentioning

confidence: 99%

High‐titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum

Cheng

Gong

et al. 2016

Biotechnology Journal

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Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw . The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA-hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.

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“…An ATP assay kit (Cominbio, China) was used to measure the ATP concentrations in the C. glutamicum/AB and C. glutamicum/Δldh-AB cells. At 3,8,16,24,28,32,40, and 48 h of batch culturing in the 5-L fermenter, cell samples were taken by centrifugation and washed once with 0.1 mM PBS (pH 7.0). All cell samples were diluted to OD 600 ¼ 1 and used for subsequent measurements per the manufacturer's protocol.…”

Section: Intracellular Adenosine Triphosphate (Atp) Concentration Meamentioning

confidence: 99%

“…[6] The original method for producing HA was to extract it from rooster combs, bovine eyes, or human umbilical cords [7] ; however, the extraction method was problematic due to its low productivity and high purification costs. In the 1980s, a fermentation method was proposed in Japan using the native HA producer, Streptococcus, [8][9][10][11] and a HA titer of 6 to 7 g L À1 was produced on an industrial scale. [10] However, the pathogenicity of the wild-type Streptococcus hindered its development, and thus nonpathogenic mutant strains were evaluated.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Biosynthesis of Hyaluronic Acid Using Engineered Corynebacterium glutamicum Via Metabolic Pathway Regulation

Cheng

Luozhong

Guo

et al. 2017

Biotechnology Journal

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Hyaluronic acid (HA) is a polysaccharide used in many industries such as medicine, surgery, cosmetics, and food. To avoid potential pathogenicity caused by its native producer, Streptococcus, efforts have been made to create a recombinant host for HA production. In this work, a GRAS (generally recognized as safe) strain, Corynebacterium glutamicum, is engineered for enhanced biosynthesis of HA via metabolic pathway regulation. Five enzymes (HasA-HasE) involved in the HA biosynthetic pathway are highlighted, and eight diverse operon combinations, including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE, are compared. HasAB and HasABC are found to be optimal for HA biosynthesis in C. glutamicum. To meet the energy demand for HA synthesis, the metabolic pathway that produces lactate is blocked by knocking out the lactate dehydrogenase (LDH) gene using single crossover homologous recombination. Engineered C. glutamicum/Δldh-AB is superior and had a significantly higher HA titer than C. glutamicum/Δldh-ABC. Batch and fed-batch cultures of C. glutamicum/Δldh-AB are performed in a 5-L fermenter. Using glucose feeding, the maximum HA titer reached 21.6 g L , more than threefolds of that of the wild-type Streptococcus. This work provides an efficient, safe, and novel recombinant HA producer, C. glutamicum/Δldh-AB, via metabolic pathway regulation.

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Production of Hyaluronic Acid by Streptococcus

Cited by 4 publications

References 4 publications

A high‐throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

A high‐throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

High‐titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum

Enhanced Biosynthesis of Hyaluronic Acid Using Engineered Corynebacterium glutamicum Via Metabolic Pathway Regulation

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