Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant

Vartak, Narendra B.; Lin, C.C.; Cleary, Joseph M.; Fagan, Matthew J.; Saier, Milton H.

doi:10.1099/13500872-141-9-2339

Cited by 39 publications

(13 citation statements)

References 29 publications

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“…Plasmid inserts in E. coli DH5a strains were identified using X-gal (40 lg/ml) and IPTG (20 lg/ml). For gellan production, cells were grown in S-60 salts medium, modified from that described previously [44], containing per liter: 0. 23 …”

Section: Methodsmentioning

confidence: 99%

“…Gene library construction S. elodea chromosomal DNA was purified [44], partially digested with PstI and fractionated by sucrose gradient centrifugation [10-40% gradient, centrifuged in an SW41 rotor at 25,000 rpm (107,000 g) for 20 h]. Fractions containing DNA fragments of approximately 20-30 kb were pooled, purified by ethanol precipitation and the isolated DNA ligated into the polylinker of pLAFR3 [41].…”

Section: Mutagenesismentioning

confidence: 99%

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Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461

Harding¹,

Patel²,

Coleman

2004

Journal of Industrial Microbiology and Biotechnology

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Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [-->4)-alpha- l-rhamnose-(1-->3)-beta- d-glucose-(1-->4)-beta- d-glucuronic acid-(1-->4)-beta- d-glucose-(1-->]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP- l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes ( gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.

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Section: Methodsmentioning

confidence: 99%

Section: Mutagenesismentioning

confidence: 99%

Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461

Harding¹,

Patel²,

Coleman

2004

Journal of Industrial Microbiology and Biotechnology

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“…Genetic studies into exopolysaccharide-producing microorganisms of industrial interest have tended to focus upon improvements in the production or functional characteristics of their EPS's (Vartak et al 1995;Martins and Sa´-Correia 1993). We have made similar studies with the mauran producer strain S-30 using strategies such as insertional mutagenesis and have produced a mutant, called TK26, which produces higher quantities of exopolysaccharide than the wild-type strain does.…”

Section: Genetic Studies Of H Mauramentioning

confidence: 95%

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest

Llamas

Moral

Martínez‐Checa

et al. 2006

Antonie Van Leeuwenhoek

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Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF's) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.

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“…2001), respectively. Second, Vartak et al. (1995) inactivated the zwf gene (encoding G6P‐dehydrogenase) by site‐specific mutagenesis to envisage diverting carbon flow towards gellan biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…To meet the growing demand of gellan gum, various traditional processes have been employed to improve the fermentative production, such as isolation of improved mutant strains (Lobas et al 1992;West 2002) and optimization of culture conditions (Jin et al 2003;West 2003;Wang et al 2006;Banik et al 2007). Vartak et al (1995) and Bower et al (2001) attempted to divert carbon flow towards gellan synthesis by constructing genetically engineered mutant strains. Subsequently, significant progresses in the genetics of gellan biosynthesis have been achieved (Sá-Correia et al 2002;Harding et al 2004;Fialho et al 2008): it is a three-step process.…”

Section: Introductionmentioning

confidence: 99%

Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production

Chen

et al. 2010

Journal of Applied Microbiology

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Aims: To improve a commercially used strain for gellan production by exogenous Vitreoscilla haemoglobin (VHb). Methods and Results: VHb gene was expressed in Sphingomonas elodea under the control of constitutive bla promoter. Biochemical activity of expressed VHb was confirmed by CO‐difference spectra analysis that exhibited a characteristic absorption maximum at 419 nm. During cultivation, not only enhanced cell growth was detected, but also 20% improvement in gellan production was observed after 48 h of incubation, with a maximum yield of 16·82 g l−1. Moreover, maximum sucrose conversion efficiency (g gellan per g sucrose) was 57·8, 20% higher than that of the parental strain. We further examined the polysaccharide production of VHb‐expressing strain at different aeration levels in Erlenmeyer flasks. Again, in all cases, a significant enhancement of gellan production was observed, and the enhancement was more significant under oxygen‐limiting conditions (up to 26·8%). Conclusions: VHb exhibited positive effect on cell growth and gellan yield of S. elodea, especially under hypoxic conditions. Significance and Impact of the Study: This is the first application of VHb as an effective metabolic engineering strategy in S. elodea to regulate cell growth and optimize gellan yield.

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Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant

Cited by 39 publications

References 29 publications

Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461

Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest

Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production

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