Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

Enhanced production of GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose by overexpression of NADPH regenerator in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.     

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Guanosine 5′-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5′-diphosphate (GDP)-l-fucose. In this study, improvement of GDP-l-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-l-fucose. The effects of overexpression of inosine 5′-monophosphate (IMP) dehydrogenase, guanosine 5′-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine–inosine kinase (Gsk) on GDP-l-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-l-fucose production. Maximum GDP-l-fucose concentration of 305.5 ± 5.3 mg l⁻¹ was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes. Such an enhancement of GDP-l-fucose production could be due to the increase in the intracellular level of GMP.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Analysing overexpression of l-valine biosynthesis genes in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum

l-Valine biosynthesis was analysed by comparing different plasmids in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum strains in order to achieve an optimal production strain. The plasmids contained different combinations of the genes ilvBNCDE encoding for the l-valine forming pathway. It was shown that overexpression of the ilvBN genes encoding acetolactate synthase is obligatory for efficient pyruvate conversion and to prevent l-alanine as a by-product. In contrast to earlier studies, overexpression of ilvE encoding transaminase B is favourable in pyruvate-dehydrogenase-negative strains. Its amplification enhanced l-valine formation and avoided extra- and intracellular accumulation of ketoisovalerate.     

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN ^r C genes at 31?°C in 72?h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15?% saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN ^r C genes, l-alanine concentration was reduced to 0.18?g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, and 0.22?g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15?g/L and 0.173?g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, 38.82?g/L and 0.252?g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.     

l-Ribulose production by an Escherichia coli harboring l-arabinose isomerase from Bacillus licheniformis

Recombinant Escherichia coli harboring the l-arabinose isomerase (BLAI) from Bacillus licheniformis was used as a biocatalyst to produce l-ribulose in the presence of borate. Effects of substrate concentration, the borate to l-arabinose ratio, pH, and temperature on the conversion of l-arabinose to l-ribulose were investigated. l-Ribulose production was efficient when pH was higher than 9 and temperature was higher than 50 °C. Borate addition to the reaction mixture was essential for high conversion of l-arabinose to l-ribulose as it resulted in an equilibrium shift in favor of the product. Under the optimal conditions determined by response surface methodology, the E. coli harboring BLAI produced 375 g l⁻¹ L-ribulose from 500 g l⁻¹ l-arabinose at a reaction time of 60 min, corresponding to a conversion yield of 75% and productivity of 375 g l⁻¹ h⁻¹. When the resting recombinant E. coli cells were recycled, 85% of the yield was obtained even after seven cycles of reuse. The productivity and final concentration of l-ribulose obtained in the present study were the highest yet reported.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Mutation-Screening in <Emphasis Type="SmallCaps">l</Emphasis>-(+)-Lactic Acid Producing Strains by Ion Implantation

In this paper, in order to obtain some industrial strains with high yield of l-(+)-lactic acid, the wild type strain Lactobacillus casei CICC6028 was mutated by nitrogen ions implantation. By study, it was found that the high positive mutation rate was obtained when the output power was 10 keV and the dose of N⁺ implantation was 50 × 2.6 × 10¹³ ions/cm². In addition, the initial screening methods were also studied, and it was found that the transparent halos method was unavailable, for some high yield strains of l-(+)-lactic acid were missed. Then a mutant strain which was named as N-2 was isolated, its optimum fermentation temperature was 40°C and the l-(+)-lactic acid yield was 136 g/l compared to the original strain whose optimum fermentation temperature was 34°C and l-(+)-lactic acid production was 98 g/l. Finally, High Performance Liquid Chromatography method was used to analyze the purity of l-(+)-lactic acid that was produced by the mutant N-2, and the result showed the main production of N-2 was l-(+)-lactic acid.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Genome shuffling and high-throughput screening of <Emphasis Type="Italic">Brevibacterium flavum</Emphasis> MDV1 for enhanced <Emphasis Type="SmallCaps">l</Emphasis>-valine production

l-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for l-valine grows rapidly, there is an increasing interest to develop an efficient l-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield l-valine strains to replace the traditional l-valine assay. l-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six l-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an l-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of l-valine (0.598 mol l-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m³ fermentor, this strain produced?>?66.8 g/L l-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of l-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

Comparative study of the enzymatic synthesis of l-valine by purified enzymes,crude extract,intact or permeabilized cells from Bacillus megaterium

Summary A detailed study on the reductive amination of -ketoisovalerate to l-valine by l-valine dehydrogenase using glucose dehydrogenase as an NADH regeneration enzyme was performed. The presence of both enzyme activities in Bacillus megaterium ATCC 39 118 permitted a direct and systematic comparison of the performances (initial l-valine production rate, productivity, molar conversion yield) of different types of conversion systems: purified enzymes or crude extract and whole cells, intact or permeabilized. A maximal l-valine productivity of 8 mmol·l^–1 · h^–1 was obtained using purified enzymes which constituted the most efficient system with a maximal rate of 0.87 mol · ml^–1 · min^–1 and a molar conversion yield of 0.91. Permeabilized cells were also an attractive system because of their easy preparation and of the good performances attained.Offprint requests to: F. Monot     

Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives

The concept of utilizing excess biomass or wastes from agricultural and agro-industrial residues to produce energy, feeds or foods, and other useful products is not necessarily new. Recently, fermentation of biomass has gained considerable attention due to the forthcoming scarcity of fossil fuels and also due to the necessity of increasing world food and feed supplies. A cost-effective viable process for lactic acid production has to be developed for which several attempts have been initiated. Fermentation techniques result in the production of either d (−) or l (+) lactic acid, or a racemic mixture of both, depending on the type of organism used. The interest in the fermentative production of lactic acid has increased due to the prospects of environmental friendliness and of using renewable resources instead of petrochemicals. Amylolytic bacteria Lactobacillus amylovorus ATCC 33622 is reported to have the efficiency of full conversion of liquefied cornstarch to lactic acid with a productivity of 20 g l⁻¹ h⁻¹. A maximum of 35 g l⁻¹ h⁻¹ was reported using a high cell density of L. helveticus (27 g l⁻¹) with a complete conversion of 55- to 60-g l⁻¹ lactose present in whey. Simultaneous saccharification and fermentation is proved to be best in the sense of high substrate concentration in lower reactor volume and low fermentation cost. In this review, a survey has been made to see how effectively the fermentation technology explored and exploited the cheaply available source materials for value addition with special emphasis on lactic acid production.     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.