Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Potential α-glucosidase inhibitors from thermal transformation of (+)-catechin

Thermal transformation of the (+)-catechin (1) with heating processing afforded a new oxidation product, gambiriin D (2), along with catechin [6′–8]-catechin (3), and (+)-epicatechin (4). The structure of a new catechin dimer with C–C linkage was determined on the basis of spectroscopic data interpretation. The catechin dimers 2 and 3 exhibited significantly improved inhibitory activities against α-glucosidase, with IC₅₀ values of 0.16 ± 0.2 and 0.14 ± 0.2 μM, respectively, when compared to parent (+)-catechin. Kinetic analysis showed that the two effective compounds 2 and 3 have noncompetitive modes of action.     

Medium optimization for the high yield production of single (+)-terrein by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca

Terrein has potential application in the fields of medicine, cosmetology and agriculture, however, the chemical synthesis of terrein with single configuration is a difficult task, and the biosynthesis of terrein always results in low production (ca. 0.33–400 mg/L). In this study, we reported an Aspergillus terreus strain PF26 which could produce (+)-terrein on a high level. After the selection of a suitable basic medium, the component concentrations were optimized using Plackett–Burman design and response surface methodology. Consequently, an optimal medium containing 28.41 g glucose, 23.18 g maltose, 20.00 g mannitol, 8.52 g malt extract, 10.00 g monosodium glutamate 10.00 g NH₄Cl in 1 L ASW was obtained, and a high (+)-terrein production of 3.71 g/L fermentation broth was achieved, which represents the highest fermentation production of (+)-terrein to date. The result highlighted the industry's potential of A. terreus strain PF26 in the production of bioactive (+)-terrein on a large-scale.     

Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis

Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) reached 722.46 mg/L and 23.16 mg/g dry cell weight (DCW), respectively, in 5.0 L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) can be produced from glycerol via two enzymatic reactions catalyzed by a coenzyme B₁₂-dependent glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) in Klebsiella pneumoniae. As the intracellular GDHt activity in K. pneumoniae is high, the overall rate of 3-HP production is controlled by the ALDH activity. To examine the effect of different ALDH activity on 3-HP production, three different ALDHs, AldH from Escherichia coli (EaldH), PuuC from K. pneumoniae (PuuC) and KGSADH from Azospirillum brasilense (KGSADH), were overexpressed and compared in various recombinant K. pneumoniae strains. In addition, the genes encoding DhaT and YqhD, which are responsible for the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PDO), were disrupted individually from K. pneumoniae to enhance the carbon flux from 3-HPA to 3-HP. When the ALDH activity was measured in various recombinant K. pneumoniae, KGSADH showed the highest crude cell activity of 8.0 U/mg protein, which was 2 and 4 times higher than that of PuuC and EaldH, respectively. The different ALDH activities had a significant effect on 3-HP production in a flask culture containing 100 mM glycerol, and K. pneumoniae ΔdhaT (KGSADH) resulted in the highest titer (64 mM) among the nine recombinant strains (three ALDH × three host strains; one wild type and two mutants). In glycerol fed-batch bioreactor cultivation, K. pneumoniae ΔdhaT (KGSADH) exhibited 3-HP production at >16 g/L in 48 h with a glycerol carbon yield of >40%. In comparison, K. pneumoniae ΔdhaT (PuuC) produced only 11 g/L 3-HP in 48 h with a yield of >23%. This study demonstrates that a high ALDH activity is essential for the effective production of 3-HP from glycerol with recombinant K. pneumoniae.     

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔC_p, with NADP binding ΔC_p closer to what is expected of crystallographic structures, while NADPH ΔC_p is three times larger. The estimated dimer–tetramer association constant is 1.5 · 10⁶ M^? 1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.     

Inhibition of human vascular NADPH oxidase by apocynin derived oligophenols

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC₅₀ = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47^phox and p22^phox. To that end, while apocynin was unable to block the interaction of his-tagged p47^phox with a surface immobilized biotinylated p22^phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC₅₀ = 1.6 μM). These results provide evidence that peroxidase-generated AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.     

Enhanced cephalotaxine production in Cephalotaxus mannii suspension cultures by combining glycometabolic regulation and elicitation

To study the combination effects of glycometabolic regulator NaF and elicitor methyl jasmonate (MJ) on cephalotaxine production in Cephalotaxus mannii suspension cultures, NaF of 10 mg/L, MJ of 100 μmol/L or both of them (NaF + MJ for short below) were added to the shake-flask cultures of C. mannii cell. It was found that NaF increased the activity of glucose 6-phosphate dehydrogenase (G6PDH), but had no significant effects on phenylalanine ammonium-lyase (PAL) activity and phenols formation. In contrast, MJ could activate PAL activity and led to phenols accumulation, but had no significant effects on G6PDH activity. To explore the effects of NaF and MJ on cephalotaxine biosynthesis, harringtonine and homoharringtonine, the two cephalotaxines, were analyzed in this work. The results obtained indicated that NaF + MJ treatment showed the strongest promotion of production in all tests. Harringtonine yield in NaF + MJ treated cells (7.245 mg/L) was 4.8-fold higher than that in control cells (1.506 mg/L), 1.7-fold that in NaF-treated cells (4.12 mg/L) and 1.6-fold that in MJ-treated cells (4.458 mg/L), respectively. No homoharringtonine was found besides in NaF + MJ treated cells (0.491 mg/L). With respect of the product release rates, they were 0%, 78%, 24% and 62% in control, NaF, MJ and NaF + MJ treatment, respectively. These results suggest that the combination of NaF and MJ had contributed to the synthesis and secretion of cephalotaxine in C. mannii cells.     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol

2-Keto-l-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from d-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five l-sorbose dehydrogenases (SDH) and two l-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to l-sorbose. The optimum combination produced 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4 g/L of 2-KLG after 168 h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2 g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

A bioprocess for the production of high concentrations of R-(+)-α-terpineol from R-(+)-limonene

A bioprocess with a high conversion rate of limonene to α-terpineol was described. The enzyme hydratase involved in this process was found as being cofactor independent, non-inducible and able to perform the transformation of both R-(+) and S-(−)-limonene. The system used consisted of a biphasic medium in which the aqueous phase contained a concentrated resting cells of Sphingobium sp. and the organic phase was sunflower oil. After 30 h at 30 °C ca. 25 g of R-(+)-α-terpineol per liter of organic phase were obtained from R-(+)-limonene in Erlenmeyer flasks. Performance of the bioconversion in a bioreactor increased the production rate with no changes in yield and maximal R-(+)-α-terpineol concentration, which demonstrated that experiments in flasks were limited by liquid–liquid transport phenomena. A mathematical model able to explain the fact that the reaction always stopped before the precursor became exhausted has also been proposed and validated. Finally, the process reported was the most promising alternative for the biotechnological production of natural R-(+)-α-terpineol published so far and up to ca. 130 g L⁻¹ metabolite could finally be obtained.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h⁻¹ and 0.6 h⁻¹. At 0.45 h⁻¹, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD⁺ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h⁻¹, 8% at D = 0.30 h⁻¹, 14% at D = 0.45 h⁻¹ and 26% for D = 0.62 h⁻¹, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.     

5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway

ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C₅ pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2 g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1 g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering.     

Modelling and simulation of continuous L (+) lactic acid production from sugarcane juice in membrane integrated hybrid-reactor system

Modelling and simulation was done for a two-stage membrane-integrated hybrid reactor system for continuous production of L (+) lactic acid under non-neutralizing conditions. The model captures microbial conversion of sugar cane juice to lactic acid under substrate–product inhibitions with downstream purification by nanofiltration. All the major phenomena and the governing parameters like fluid flow, feed dilution, substrate–product inhibitions, Donnan and steric effects during micro and nanofiltration for cell recycle, product separation and purification have been reflected in the modelling. The model describes a green, integrated continuous process of direct lactic acid production starting with a cheap, renewable carbon source. The highest lactic acid concentration achieved after the final stage of nanofiltration was 66.97 g/L at 13 kg/cm² operating pressure when the overall productivity reached 12.40 g/(L h). The developed model could successfully predict production, purification and transport of lactic acid through two stage membrane modules. Performance of the model was very good as indicated in the high overall correlation coefficient (R² > 0.980) and the low relative error (RE < 0.1).     

Optimizing l-(+)-lactic acid production by thermophile Lactobacillus plantarum As.1.3 using alternative nitrogen sources with response surface method

The effects of five alternative nitrogen sources, namely, malt sprout (MS), corn steep liquor (CSL), NH₄Cl, NH₄NO₃ and diamine citrate (DC) were investigated on the l-(+)-lactic acid (LA) production by thermophile Lactobacillus plantarum As.1.3. Through the statistical analysis of the results by three steps of response surface methodology (RSM) design, MS and CSL were found to have significant effects on the LA production and their optimal concentrations in the medium should be 16.0 g/L and 12.0 g/L, respectively. The verification of the optimized medium showed that the maximum specific growth rate (μ_m) was 1.09 h⁻¹, the cell yield coefficient (Y_X/S) and the l-(+)-lactic acid yield coefficient (Y_P/S) were 0.233 (OD₆₂₀/g) and 0.98 (g/g), and the maximum volumetric productivity and the average volumetric productivity were 13.0 g/L h and 3.20 g/L h, respectively. The results indicate that the LA production can also be enhanced with the inexpensive nitrogen source alternatives.     

A straightforward procedure to biosynthesise melatonin using freshly chopped Achillea millefolium L. as reagent

d, l-Tryptophan as the precursor of melatonin and Achillea millefolium L. as the plant cell tissue were used in order to force the plant enzymatic system to enhance the production of melatonin.The biotransformation protocol was performed by adding the precursor to the freshly chopped plant material suspended in water and stirred at room temperature in total darkness. The melatonin content was evaluated by LC-DAD-ESI-MS. The precursor-treated sample gave at least six times (345 ng/g_{fresh plant}) the melatonin amount usually produced in young plants of A. millefolium L. (50 ng/g_fresh plant).The present study shows that, in principle, adding a specific precursor to a suitable freshly chopped plant material can significantly enhance a secondary metabolite production.