Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

Glutathione: a review on biotechnological production

This Mini-Review summarizes the historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years. Glutathione is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (l-glutamic acid, l-cysteine, glycine). Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material. In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale. At the molecular level, the genes gshA and gshB, which encode the enzymes -glutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E. coli, S. cerevisiae, and Lactococcus lactis. It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.     

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway

Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l⁻¹ in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l⁻¹). Production was further boosted to 0.48 g l⁻¹ when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l⁻¹). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast.     

Enhanced glutathione production by using low-pH stress coupled with cysteine addition in the treatment of high cell density culture of Candida utilis

Aims: To investigate the effects of pH stress coupled with cysteine addition on glutathione (GSH) production in the treatment of high cell density culture of Candida utilis. Methods and Results: We have previously observed that most Candida utilis cells remained viable after being subjected to pH at 1·2 for 3 h and that some intracellular GSH leaked into the medium. A cysteine addition strategy was applied in fed‐batch production of GSH. A single cysteine addition resulted in higher GSH yield than two separate additions without pH stress. An increase in intracellular GSH content triggered inhibition of γ‐glutamylcysteine synthetase (γ‐GCS). A strategy that combines cysteine addition with low‐pH stress was developed to relieve the inhibition of γ‐GCS. Conclusion: Without pH stress, single shot and double shot cysteine addition yielded a total GSH of 1423 and 1325 mg l^?1. In comparison, a low‐pH stress counterpart resulted in a total GSH of 1542 and 1730 mg l^?1, respectively. With low‐pH stress, we observed GSH secretion into the medium at 673 and 558 mg l^?1 and an increase in the γ‐GCS activity by 1·2‐ and 1·5‐fold, respectively. The specific GSH production yield increased from 1·76% to 1·91% (w/w) for single shot, and 1·64% to 2·14% for double shots. Significance and Impact of the Study: Low‐pH shift was applied to alleviate the feedback inhibition of intracellular GSH on γ‐GCS activity by secreting GSH into the medium. This strategy is coupled with cysteine addition to enhance GSH production in Candida utilis.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Lincomycin,rational selection of high producing strain and improved fermentation by amino acids supplementation

Based on the report that the introduction of the biosynthetic precursor of lincomycin, propylproline, could increase the production of lincomycin (Bruce et al. in US Patent 3,753,859, 1973), a mutant strain pro10–20, with resistance of feedback suppression of proline (an analog of propylproline) was thus selected and lincomycin production increased by 10%. The addition of three amino acids (l-proline, l-tyrosine, l-alanine) which are the precursors of propylproline to the fermentation medium was found to enhance the accumulation of l-dopa through different pathways and was favorable to lincomycin biosynthesis. The production of lincomycin was increased by 23, 10, 13%, respectively, with the addition of 0.05 g L⁻¹ l-proline at 60 h, 0.005 g L⁻¹ l-tyrosine and 0.1 g L⁻¹ l-alanine directly in the medium.     

Increased synthesis of α‐tocopherol,paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

Aims: To analyse the production of different metabolites by dark‐grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l^?1), malate (2 g l^?1) and ethanol (10 ml l^?1) (GM + EtOH); glutamate (7·15 g l^?1) and ethanol (10 ml l^?1); or malate (8·16 g l^?1), glucose (10·6 g l^?1) and NH₄Cl (1·8 g l^?1) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6‐fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α‐tocopherol after 120 h identified by LC‐MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)]^?1, respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)^?1. For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP‐HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark‐grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α‐tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α‐tocopherol and some amino acids. The concentrations of α‐tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio‐molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.     

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

Summary As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn²⁺ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.Abbreviation POD peroxidase     

Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose

Aims: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source. Methods and Results: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2·8 and 3 g l⁻¹ h⁻¹ in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l⁻¹. Conclusions: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content. Significance and Impact of the Study: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.     

Effect of amino acids addition and feedback control strategies on the high-cell-density cultivation of Saccharomyces cerevisiae for glutathione production

Controlling glucose feeding rate, according to the change of ethanol concentration and respiratory quotient (RQ), has been studied in high-cell-density cultivation of Saccharomyces cerevisiae for glutathione (GSH). GSH yield and dry cell weight reached 1620 mg/L and 140 g/L, respectively, after 52 h of cultivation. In addition, optimized single shot addition of precursor amino acids (performed in both flask and fermentor experiments) at 32 h could result in the GSH yield reached 2020 mg/L after 38 h of cultivation. The yield and productivity of GSH increased 25% and 70%, respectively. Moreover, cultivation time was reduced to 38 h, compared with results without adding the precursor amino acids.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Production of l-Leucine by a Mutant of Brevibacterium lactofermentum 2256

A potent l-leucine producer was screened among mutants of glutamic acid producing bacteria. This strain, No. 218, is one of 2-thiazolealanine resistant mutants derived from a methionine isoleucine double auxotroph of Brevibacterium lactofermentum 2256 by nitroso-guanidine.

Strain No. 218 produced 19 mg/ml of l-leucine after 72 hr cultivation when 8 % glucose and 4 % ammonium sulfate were supplied as a carbon and a nitrogen source, respectively, thus giving the yield of 23.1 % from glucose.

The addition of Fe²⁺ and Mn²⁺ in combination gave much more productivity than that of Fe²⁺ or Mn²⁺ alone.

Effects of amino acids, nucleic acids, vitamins, and the other nutrients on l-leucine production were investigated.

The fermentation product was isolated and purified from the culture, and identified as l-leucine.     

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.     

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l⁻¹ 5-KF were formed from 150 g l⁻¹ fructose within 23 h, corresponding to a space-time yield of 5.6 g l⁻¹ h⁻¹. Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Effect of Ca2+ on the retention of glycerol and amino acids in Dunaliella tertiolecta

When D. tertiolecta cells, previously incubated in a 0.5 kmol m⁻³ NaCl medium with 1mol m⁻³ Ca²⁺, were transferred to an isotonic NaCl medium without Ca²⁺, the intracellular glycerol, as well as intracellular amino acids, was transiently lost to the medium within 30 min. The transient leakage of glycerol and amino acids was enhanced by the addition of EGTA (1 mol m⁻³), while the addition of SrCl₂ (1 mol m ⁻³) or polyamines such as spermidine (5 mol m⁻³) and spermine (5 mol m⁻³) restrained the leakage caused by the lack of external Ca²⁺ of intracellular glycerol and amino acids.     

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.