Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

Isolation and characteristics of a short rod-shaped mutant of Pseudomonas aeruginosa

Abstract Among 40 short rod-shaped mutants of Pseudomonas aeruginosa PAO isolated after nitrosoguanidine mutagenesis, a stable clone designated KG1034 was obtained and studied for its cellular and genetical features. Cells of the parent strain, PAO2302 had a mean cell length of 1.9 μm, whereas that of KG1034 was 1.3 μm. The doubling time of KG1034 was less than that of the parent strain, although both strains elongated at the same rate and exhibited the same relative amounts and pattern of penicillin-binding proteins. These results suggested that the phenotype of KG1034 was due to the initiation of septation at an earlier stage. The new gene responsible for this phenotype was named srs (abbreviation for short r od shape) and mapped by conjugation between cys -54 and puuC .     

Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

A mutant strain of Leuconostoc mesenteroides B-1355 producing a glucosyltransferase synthesizing α(1→2) glucosidic linkages

A mutant strain (R1510) of Leuconostoc mesenteroides B-1355 was isolated which synthesized primarily an insoluble polysaccharide and little soluble polysaccharide when grown in sucrose-containing medium. Glucose or sucrose cultures of this strain produced a single intense band of GTF-1 activity of 240 kDa on SDS gels, and a number of faint, smaller bands. Oligosaccharides synthesized by strain R1510 from methyl-α-D-glucoside and sucrose included a trisaccharide whose structure contained an α(1→2) glucosidic linkage. This type of linkage has not been seen before in any products from strain B-1355 or its mutant derivatives. The structure of the purified trisaccharide was confirmed by ¹³C-nuclear magnetic resonance. The insoluble polysaccharide also contained α(1→2) branch linkages, as determined by methylation analysis, showing that synthesis of the linkages was not peculiar to methyl-α-D-glucoside. GTF-1, which had been excised with a razor blade from an SDS gel of a culture of the parent strain B-1355, produced the same trisaccharides as strain R1510, showing that GTF-1 from the wild-type strain was the same as GTF-1 from strain R1510. Mutant strains resembling strain R1510, but producing a single intense band of alternansucrase (200 kDa) instead of GTF-1 were also isolated, suggesting that mutations may be generated which diminished the activities for any two of the three GTFs of strain B1355 relative to the third. Strain R1554 produced a soluble form of alternansucrase, while strain R1588 produced a cell-associated form. The mechanism(s) by which specific GTFs become associated with the cells of L. mesenteroides was not explored. Received 12 May 1998/ Accepted in revised form 16 July 1998     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Isolation and characterization of an aluminum-sensitive mutant of Pseudomonas fluorescens

An aluminum-sensitive mutant was isolated by chemical mutagenesis using 1-methyl-3-nitro-1-nitrosoguanidine. Although this mutant of Pseudomonas fluorescens was susceptible to aluminum, it did respond in a manner analogous to the wild-type when challenged with either Ca²⁺, or Ga³⁺ or Zn²⁺. However, the aluminum-sensitive mutant did not grow in media with iron. Calcium detoxification appeared to be attained by the elaboration of calcite, a detoxification strategy observed in the wild-type Pseudomonas fluorescens. There was a 40% diminution in biomass when the mutant was cultured in a control medium as compared to the wild-type. The aluminum-sensitive mutant had a characteristic yellow coloration. Electrophoretic analyses of the soluble cellular extract revealed numerous variations between the aluminum-sensitive mutant and wild-type Pseudomonas fluorescens. Bands corresponding to approximate molecular masses of 158 kDa, 116 kDa, 97 kDa, 55 kDa, and 19 kDa were absent in the aluminum-sensitive mutant. Furthermore, bands attributable to molecular masses of 90 kDa and 15 kDa were only evident in the aluminum-sensitive Pseudomonas. It appears that the chemical mutagenesis may have affected only aluminum and iron(III) metabolism in this organism.     

Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461

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)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--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.     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6.

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Bioaugmentation of a methyl parathion contaminated soil with Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (MP) and para-nitrophenol as the sole source of carbon, nitrogen and energy. In this study, strain WBC-3 was inoculated into lab-scale MP-contaminated soil for bioaugmentation. Accelerated removal of MP was achieved in bioaugmentation treatment compared to non-bioaugmentation treatment, with complete removal of 0.536 mg g⁻¹ dry soil in bioaugmentation treatment within 15 days and without accumulation of toxic intermediates. The analysis of denaturing gradient gel electrophoresis and real-time PCR showed that strain WBC-3 existed stably during the entire bioaugmentation period. Simultaneously, redundancy analysis for evaluating the relationships between the environmental factors and microbial community structure indicated that the indigenous bacterial community structure was significantly influenced by strain WBC-3 inoculation (P = 0.002).     

Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol

Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas.     

Use of whey for production of exocellular polysaccharide by a mutant strain ofXanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac ⁺ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium. Translated by Č. Novotny     

Isolation of a petite mutant from a histidine auxotroph of Candida albicans and its characterization

Respiration-deficient (petite) mutations have been induced in various yeasts, which are categorized as petite-positive. Candida albicans was classified among the petite-negative yeasts. Since then, a few reports have appeared, describing the isolation of petite mutants in C. albicans. We report in the present study on the isolation of a petite mutant of C. albicans-SAR1. This mutant was isolated from a histidine auxotroph of C. albicans after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, thus our petite mutant carries a double mutation. SAR1 was characterized morphologically, biochemically and ultrastructurally. The results revealed differences from the wild type in respect to morphological, physiological and biochemical characteristics. Electron microscopy showed that the cells of the petite mutant contain only very few mitochondria that looked ‘thread like’ without any cristae. The significance of the mutation in the virulence of the mutant vs. that of the wild-type is being assessed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of a transglucosidase-nonproducing mutant ofAspergillus awamori yielding improved quality and production of amyloglucosidase preparations

Summary The fermentative production of amyloglucosidase (AMG) by differentAspergillus species simultaneously yields transglucosidase (TG), which is undersirable in the conversion of starch to dextrose, as it catalyses the reversion of dextrose and maltose to maltosaccharides, in turn providing disproportionately lower yields of dextrose (DX). To overcome this problem, using UV-irradiation, a novel mutant (ND-1-283) has been isolated fromAspergillus awamori, which has lost the ability to produce TG and which secretes 45% more AMG than its parent strain, giving the mutant a dual operational advantage. The inability of this mutant to produce TG was demonstrated by thin layer chromatography (TLC) of starch hydrolysate; this was substantiated by obtaining 96.0% DX (w/w) at the end of saccharification.     

Isolation of a new thermohalophilic Thermus thermophilus strain from hot spring, able to grow on a renewable source of polysaccharide

A thermohalophilic strain, Samu-Sal, isolated from hot springs of the Mount Grillo (Baia, Naples, Italy) at a depth of 60 m, according to its genotypic analyses is related to Thermus genus and should be classified as a new strain of Thermus thermophilus. Strain Samu-SA1 grew using, as sole carbon source, a polysaccharide extracted from waste industrial tomato process with a yield of 3.5 g l(-1). Strain Samu-SA1 synthesized several alpha- and beta-glycosidases.     

产壳聚糖酶菌株的分离、鉴定及发酵条件优化

从废弃食用菌培养基周围土壤中分离得到一株产壳聚糖酶的菌株,结合形态学特征与26SrDNA序列进行了分类学鉴定,结果表明,该菌株与高山被孢霉(Mortierella alpina)的同源性较高,达99%,初步鉴定为被孢霉属的一种,命名为KB-1001。并对该菌株的产酶特性进行了研究,结果表明,该菌株液体发酵培养产酶高峰出现在第84h,最适碳源为1%的水溶性壳聚糖,最适氮源为1.87%的(NH4)2SO4,摇瓶培养的最适初始pH值为6.0,最适温度为28℃～30℃,接种量为4%,最佳装瓶量为70 mL/250 mL,150 r/min摇瓶培养,经优化培养后,该菌株发酵液中壳聚糖酶活力最高达到8.130 U/mL。比原始的未经发酵条件优化的产酶活性提高了12.78%。     

一株新型高产PHB假单胞菌SCH17的分离和特征分析

通过丁醇富集筛选,从土壤样品中筛选到一株菌株SCH17。经过生理特性和16S rRNA分析,鉴定菌株SCH17属于假单胞菌属。透射电镜显示该菌细胞内聚集了大量颗粒状物质,经过氯仿抽提和核磁共振分析,确认这些颗粒物质是聚β-羟基丁酸(PHB)。通过对碳源和氮源的优化,得到最佳积累PHB的碳源是果糖,氮源是蛋白胨。在该培养基中仅需发酵14 h,菌体干重和PHB含量均达到最大,分别为3.52 g/L和2.69 g/L,PHB含量高达细胞干重的76%。