Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium

d-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO₂ as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L⁻¹ and a production rate of 0.15 g mannitol L⁻¹ day⁻¹. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO₂ in cyanobacteria.     

Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. De novo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L⁻¹ AVA in a fed-batch process and achieved a high yield of 0.21 g g⁻¹. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L⁻¹ AVA at a yield of 0.34 g g⁻¹ and a maximum productivity of 1.52 g L⁻¹ h⁻¹, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers     

Purification and characterization of a Na+/K+ dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min⁻¹ ml⁻¹ of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min⁻¹ ml⁻¹ at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients (K_m)app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V_max, for immobilized microsomes is calculated to be 322 nmoles min⁻¹ ml⁻¹ compared with 145 nmols min⁻¹ ml⁻¹ for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.     

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg⁻¹ and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l⁻¹), a molar yield of 0.4 mol mol⁻¹, and a volumetric productivity of 2.1 mmol l⁻¹ h⁻¹.     

From zero to hero – Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2 g L⁻¹ h⁻¹, and a final titer of 88 g L⁻¹. The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.     

Evaluation of fermentation waste (Corynebacterium glutamicum) as a biosorbent for the treatment of nickel(II)-bearing solutions

This research highlights the possibility of employing a fermentation industry waste (Corynebacterium glutamicum) for the removal of nickel(II) ions from aqueous solution. Furthermore, it necessitates the importance of detailed examinations on the possible differences in the biosorption performance, even for the same biomass, but from different origins. Two types of C. glutamicum, obtained from different industrial sources, were used in this study. With respect to nickel speciation and biosorption performance, pH 6 was identified as an optimal condition. Of the two types of C. glutamicum used, the biomass with excess negatively charged groups performed well in the binding of Ni²⁺ ions. To enhance the feasibility of using the biomass in column mode, as well as its reuse for multiple cycles, C. glutamicum was immobilized in a polysulfone matrix. Both the free and immobilized biomasses performed relatively well, with maximum experimental uptakes of 111.4 and 102.4 mg g⁻¹, respectively. An up-flow packed column loaded with immobilized biomass was employed for the removal of Ni²⁺ ions. The column performed well in the biosorption of nickel(II), and exhibited a delayed and favorable breakthrough curve, with Ni²⁺ uptake and percentage removal of 48.1 mg g⁻¹ biomass and 60.4%, respectively.     

The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization

Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Treatment of dye-rich wastewater by an immobilized thermophilic cyanobacterial strain: Phormidium sp.

The removal of Remazol Blue and Reactive Black B by the immobilized thermophilic cyanobacterial strain Phormidium sp. was investigated under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye removal. In the experiments, performed at pH 8.5, with different initial dye concentrations between 9.1 mg l⁻¹ and 82.1 mg l⁻¹ and at 45 °C, calcium alginate immobilized Phormidium sp. showed high dye decolorization, with maximum uptake yields ranging from 50% to 88% at all dye concentrations tested. When the effects of high dye concentrations on dye removal were investigated, the highest uptake yield in the beads was 50.3% for 82.1 mg l⁻¹ Remazol Blue and 60.0% for 79.5 mg l⁻¹ Reactive Black B. The highest color removal was detected at 45 °C and 50 °C incubation temperatures for all dye concentrations. As the temperature decreased, the removal yield of immobilized Phormidium sp. also decreased. At about 75 mg l⁻¹ initial dye concentrations, the highest specific dye uptake measured was 41.29–41.17 mg g⁻¹ for Remazol Blue and 47.69–43.82 mg g⁻¹ for Reactive Black B at 45 °C and 50 °C incubation temperatures, respectively, after 8 days incubation.     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation

Wild-type Corynebacterium glutamicum produced 0.6 g l⁻¹ xylitol from xylose at a productivity of 0.01 g l⁻¹ h⁻¹ under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l⁻¹ xylitol at 3.1 g l⁻¹ h⁻¹. To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS^fru) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l⁻¹ xylitol at 7.9 g l⁻¹ h⁻¹, representing the highest bacterial xylitol productivity reported to date.     

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L⁻¹), the main ethanol producers were Scheffersomyces stipitis (14.08 g L⁻¹), Scheffersomyces sp. (7.94 g L⁻¹) and Spathaspora boniae (7.16 g L⁻¹). Sc. stipitis showed the highest ethanol yield (0.42 g g⁻¹) and the highest productivity (0.39 g L⁻¹h⁻¹). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g⁻¹, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg⁻¹) and Saitozyma podzolica (0.384 U mg⁻¹). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.     

Synthesis of S-licarbazepine by asymmetric reduction of oxcarbazepine with Saccharomyces cerevisiae CGMCC No. 2266

S-licarbazepine was synthesized by asymmetric reduction of oxcarbazepine with CGMCC No. 2266. The optimum batch reduction conditions were found to consist of a reaction time of 36 h, temperature of 30 °C, and initial pH value of 7.0. The optimum concentration of the glucose co-substrate was found to be 0.3 mol L⁻¹. The addition of glucose contributed to in situ regeneration of NADPH in cells and improved conversion. Conversion increased with the addition of more biomass and with a decrease in the initial concentration of substrate. Within the membrane reactor, a continuous reduction process was used to improve production efficiency and reduce the inhibition of high-concentration substrate upon reduction. The optimum flux was found to be 20 ml h⁻¹. S-licarbazepine yield was 3.7678 mmol L⁻¹ d⁻¹ in continuous reduction over four days. The enantiometric excess of S-licarbazepine was 100% for both batch and continuous reduction processes.     

Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum

The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum.     

Immobilization of Lactobacillus rhamnosus in polyvinyl alcohol/calcium alginate matrix for production of lactic acid

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

     

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q_max) of Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate for the GFP was 1,42,860 FU ml⁻¹ and 18,000 FU ml⁻¹, respectively. The expanded bed column chromatography using Ni²⁺ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni²⁺ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.