Production of arabitol from glycerol: strain screening and study of factors affecting production yield

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.     

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l ‐phosphoserine to l ‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC_R42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l ‐homoserine is used as the substrate, while its activity toward the natural substrate l ‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerC_R42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L^?1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.     

Increasing l‐isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two‐component export system BrnFE

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Engineered Saccharomyces cerevisiae that produces 1,3‐propanediol from d‐glucose

Aims: Saccharomyces cerevisiae is a safe micro‐organism used in fermentation industry. 1,3‐Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3‐propanediol at low cost. Methods and Results: By using d ‐glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3‐propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3‐propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303‐1A by Agrobacterium tumefaciens‐mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3‐propanediol from d ‐glucose. Conclusion: Saccharomyces cerevisiae can be engineered to produce 1,3‐propanediol from low‐cost feedstock d ‐glucose. Significance and Impact of the Study: To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3‐propanediol by using A. tumefaciens‐mediated transformation. This study might lead to a safe and cost‐efficient method for industrial production of 1,3‐propanediol.     

Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l‐lactate

Campylobacter jejuni, a major food‐borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and l ‐ and d ‐lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on l ‐lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, l ‐lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD‐independent l ‐LDHs; a non‐flavin iron–sulfur containing three subunit membrane‐associated enzyme (Cj0075c‐73c), and a flavin and iron–sulfur containing membrane‐associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on l ‐lactate, as single mutants in each system grew as well as wild‐type on this substrate, while a cj0075c cj1585c double mutant showed no l ‐lactate oxidase activity and did not utilize or grow on l ‐lactate; d ‐lactate‐dependent growth was unaffected. Orthologues of Cj0075c‐73c (LldEFG/LutABC) and Cj1585c (Dld‐II) were recently shown to represent two novel families of l ‐ and d ‐lactate oxidases; this is the first report of a bacterium where both enzymes are involved in l ‐lactate utilization only. The cj0075c‐73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for l ‐lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use l ‐ and d ‐lactate as both electron‐donor and carbon source.     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Optimization of l‐DOPA production by Brevundimonas sp. SGJ using response surface methodology

l ‐DOPA (3,4‐dihydroxyphenyl‐l ‐alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing l ‐DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett–Burman design was used for screening of critical components, while further optimization was carried out using the Box–Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l^?1 tryptone, 4.207 g l^?1 l ‐tyrosine and 0.0369 g l^?1 CuSO₄, which resulted in highest l ‐DOPA yield of 3.359 g l^?1. The optimization of medium using RSM resulted in a 8.355‐fold increase in the yield of l ‐DOPA. The anova showed a significant R² value (0.9667), model F‐value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg^?1 at the 18th hour of the incubation period with dry cell weight of 0.711 g l^?1. l ‐DOPA production was confirmed by HPTLC, HPLC and GC‐MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of l ‐DOPA.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Glycerol and arabitol production by an intergeneric hybrid,PB2, obtained by protoplast fusion between Saccharomyces cerevisiae and Torulaspora delbrueckii

An intergeneric osmotolerant hybrid yeast, PB2, was used together with the parental strains to study glycerol and arabitol production in batch culture. This fusion product was previously obtained by protoplast fusion between Torulaspora delbrueckii and Saccharomyces cerevisiae. Polyols and biomass production were determined in batch culture under aerobic conditions. Under the conditions tested, using PB2 hybrid and both parental strains, the best results were obtained with the hybrid. Arabitol reached a final concentration of 70 g/l and glycerol was increased to up to 50 g/l. Electronic Publication     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Characterization of α‐rhamnosidase activity from a Patagonian Pichia guilliermondii wine strain

Aims: The purpose of this study was to characterize the α‐l ‐rhamnosidase of Pichia guilliermondii NPCC1053 indigenous wine strain from North‐Patagonian region. Methods and Results: The optimization of yeast culture conditions was carried out and the effects of oenological parameters on α‐l ‐rhamnosidase activity were evaluated. Additionally, the effect of direct contact with must and wine on α‐l ‐rhamnosidase activity was assayed. This strain showed an intracellular inducible α‐l ‐rhamnosidase activity. This enzyme was active at pH, glucose and SO₂ concentrations usually found at the beginning of the fermentation as well as retained high levels of activity after 24 h of incubation in must. Furthermore, P. guilliermondiiα‐l ‐rhamnosidase was able to release monoterpenols and alcohols from grape glycosidic extracts. Conclusions: The α‐l ‐rhamnosidase belonging to P. guilliermondii indigenous wine yeast strain showed mainly an intracellular location and evidenced interesting oenological characteristics. Significance and Impact of the Study: This study contributes to the knowledge of α‐l ‐rhamnosidases from yeast origin because at present, there are few reports about this enzymatic activity in these micro‐organisms. In addition, this work is relevant to the regional wine industry considering that this enzyme could be used in the production of more aromatic young wines.     

Catabolism of l–methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit

Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and other volatiles. l –methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C‐ and ²H‐labeled l –methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l ‐methionine aminotransferase and preserves the main carbon skeleton of l ‐methionine. The second route apparently involves the action of an l ‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l ‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l ‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l –isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l ‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l ‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l ‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.