Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

The anoxic electrode-driven fructose catabolism of Pseudomonas putida KT2440

Pseudomonas putida (P. putida) is a microorganism of interest for various industrial processes, yet its strictly aerobic nature limits application. Despite previous attempts to adapt P. putida to anoxic conditions via genetic engineering or the use of a bioelectrochemical system (BES), the problem of energy shortage and internal redox imbalance persists. In this work, we aimed to provide the cytoplasmic metabolism with different monosaccharides, other than glucose, and explored the physiological response in P. putida KT2440 during bioelectrochemical cultivation. The periplasmic oxidation cascade was found to be able to oxidize a wide range of aldoses to their corresponding (keto-)aldonates. Unexpectedly, isomerization of the ketose fructose to mannose also enabled oxidation by glucose dehydrogenase, a new pathway uncovered for fructose metabolism in P. putida KT2440 in BES. Besides the isomerization, the remainder of fructose was imported into the cytoplasm and metabolized. This resulted in a higher NADPH/NADP⁺ ratio, compared to glucose. Comparative proteomics further revealed the upregulation of proteins in the lower central carbon metabolism during the experiment. These findings highlight that the choice of a substrate in BES can target cytosolic and periplasmic oxidation pathways, and that electrode-driven redox balancing can drive these pathways in P. putida under anaerobic conditions.     

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l⁻¹ in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l⁻¹ in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l⁻¹ containing 65% PHA at the end of a 25-h incubation.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Production of fructose from inulin using mixed inulinases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

Two new effective microbial producers of inulinases were isolated from Jerusalem artichoke tubers grown in Thailand and identified as Aspergillus niger TISTR 3570 and Candida guilliermondii TISTR 5844. The inulinases produced by both these microorganisms were appropriate for hydrolysing inulin to fructose as the principal product. An initial inulin concentration of ∼100 g l⁻¹ and the enzyme concentration of 0.2 U g⁻¹ of substrate, yielded 37.5 g l⁻¹ of fructose in 20 h at 40°C when A. niger TISTR 3570 inulinase was the biocatalyst. The yield of fructose on inulin was 0.39 g g⁻¹. Under identical conditions, the yeast inulinase afforded 35.3 g l⁻¹ of fructose in 25 h. The fructose yield was 0.35 g g⁻¹ of substrate. The fructose productivities were 1.9 g l⁻¹ h⁻¹ and 1.4 g l⁻¹ h⁻¹ for the mold and yeast enzymes, respectively. After 20 h of reaction, the mold enzyme hydrolysate contained 53% fructose and more than 41% of initial inulin had been hydrolysed. Using the yeast enzymes, the hydrolysate contained nearly 38% fructose at 25 h and nearly 36% of initial inulin had been hydrolysed. The A. niger TISTR 3570 inulinases exhibited both endo-inulinase and exo-inulinase activities. In contrast, the yeast inulinases displayed mainly exo-inulinase activity. The mold and yeast crude inulinases mixed in the activity ratio of 5:1 proved superior to individual crude inulinases in hydrolysing inulin to fructose. The enzyme mixture provided a better combination of endo- and exo-inulinase activities than did the crude extracts of either the mold or the yeast individually.     

Optimization of the solvent-tolerant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 as host for the production of <Emphasis Type="Italic">p</Emphasis>-coumarate from glucose

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.     

Synthesis of aromatic amino acids from 2G lignocellulosic substrates

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner–Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l⁻¹ of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

<Emphasis Type="Italic">Narcissus</Emphasis> bulblet formation <Emphasis Type="Italic">in vitro</Emphasis>: effects of carbohydrate type and osmolarity of the culture medium

Shoot clump cultures of Narcissus cultivars St. Keverne and Hawera were used to investigate the effects of culture medium carbon supply, type of carbohydrate and osmolarity on in vitro bulblet development. Increasing the medium osmolarity using mannitol or sorbitol, which did not act as substrates for growth, failed to stimulate bulblet formation with either cultivar. An exception to this was a relatively small increase in total bulblet dry weight per culture, in the cultivar Hawera only, caused by adding 30 g l ^–1 sorbitol in combination with 30 g l^–1 sucrose. Simultaneously increasing the medium osmolarity and carbon supply using the metabolisable carbohydrate sources, sucrose, glucose, fructose or an equimolar mixture of glucose and fructose stimulated bulblet production, total dry matter accumulation and partitioning into bulblets. At comparable levels of carbon supply up to 19.0 g l^–1, bulblet development of both cultivars was similar with monosaccharide and sucrose media. This indicates that substrate supply is more important for bulblet development than osmolarity of the culture medium. The cultivar Hawera also showed similar responses to monosaccharide and sucrose media supplying 37.9 g C l^–1, despite the high osmolarity of monosaccharide media (c. 650 m Osm kg^–1, equivalent to –1.6 MPa, compared to 380 m Osm kg^–1 for sucrose medium). However in St. Keverne total dry matter accumulation and dry weight per bulblet were further stimulated only by increasing the sucrose supply from 19.0 to 37.9 g C l^–1, not by increasing the monosaccharide supply. Implications of the findings for Narcissus micropropagation are discussed.     

Fructose production from sucrose and high fructose syrup: A mycelial fungal system

Summary The use of Mucor sp. M105 and Fusarium sp. F5 in the production of fructose from sugarcane sucrose and high fructose syrup (HFS) was investigated. Although Mucor sp. could not utilize sucrose as the sole carbon and energy source for cell growth, Mucor sp. preferentially utilized glucose in a glucose:fructose (1:1) mixture during fermentation to ethanol. In contrast, Fusarium sp. utilized sucrose as sole carbon source by secretion of extracellular hydrolytic enzymes that degraded the disaccharide. In Fusarium sp., glucose formation in the medium was faster than fructose. Due to the low consumption rate of fructose, this substrate remained in the fermentation broth. The application of these biological systems for the production of fructose from either sucrose or HFS is discussed.     

Lactic acid production using two food processing wastes,canned pineapple syrup and grape invertase,as substrate and enzyme

Canned pineapple syrup, a food processing waste, was utilized as a substrate for lactic acid production by Lactococcus lactis. To improve the utilization of sucrose from the syrup, grape invertase from grape juice derived from wine production was used for sucrose hydrolysis. The highest lactic acid concentrations achieved were 20 and 92 g l^–1 from 20 and 100 g total sugars l^–1, respectively, without a lag period for sucrose consumption.     

Catalytic and biocatalytic oxidation of glucose to gluconic acid in a modified three-phase reactor

A comparative study of catalytic and biocatalytic glucose oxidation was carried out. Gluconobacter oxydans NBIMCC 1043 strain was used for biocatalytic glucose conversion. In the case of cell recycle coupled with cross-flow microfiltration the productivity and biomass concentration reached 40% and 3 g l^–1 respectively, in comparison to those of batch fermentation (21% and 2.3 g l^–1, respectively).     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l⁻¹ in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l⁻¹ were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l⁻¹ of tyrosol and 26.55 ± 0.43 g l⁻¹ of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.     

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

Summary Two mutants, unable to utilize fructose (Fru^–) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru^– mutants among survivors of mutagenesis was 1 in 10⁴. The two Fru^– mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.     

Influence of glucose,fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Batch cultivations of l-lysine-producing Corynebacterium glutamicum ATCC 21253 were carried out on the different carbon sources, glucose, sucrose and fructose. The time profiles of substrate and product concentrations were evaluated to compare kinetics and stoichiometry of lysine production. The lysine yield (mol C/mol C) on glucose was 8% higher than on sucrose and 30% higher than on fructose. The highest final biomass concentration of 5.0 g/l was obtained on glucose, whereas fructose and sucrose yielded 20% less biomass. Compared to glucose, fructose resulted in significantly higher respiration rates, a higher substrate uptake rate but a lower lysine production rate during the cultivation process. This was probably due to a higher tricarboxylic cycle activity combined with a lower activity of the pentose phosphate pathway. On sucrose, specific rates and yields differed significantly from those on fructose and glucose. Transport and metabolism of sucrose, therefore, are not a simple superposition of its building blocks, glucose and fructose. Journal of Industrial Microbiology & Biotechnology (2002) 28, 338–343 DOI: 10.1038/sj/jim/7000252 Received 28 November 2001/ Accepted in revised form 06 March 2002