Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system.

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans.

Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236T . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC3.5.1.14), designated SgAA, and an ε-lysine acylase (EC3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-l-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine.

Activities of Family 18 Chitinases on Amorphous Regenerated Chitin Thin Films and Dissolved Chitin Oligosaccharides: Comparison with Family 19 Chitinases.

Changes in mass and viscoelasticity of chitin layers in fungal cell walls during chitinase attack are vital for understanding bacterial invasion of and human defense against fungi. In this work, regenerated chitin (RChitin) thin films mimicked the fungal chitin layers and facilitated studies of degradation by family 18 chitinases from Trichoderma viride (T. viride) and family 19 chitinases from Streptomyces griseus (S. griseus) that possessed chitin-binding domains (CBDs) that were absent in the family 18 chitinases. Degradation was monitored via a quartz crystal microbalance with dissipation monitoring (QCM-D) in real time at various pH and temperatures. Compared to substrates of colloidal chitin or dissolved chitin derivatives and analogues, the degradation of RChitin films was deeply affected by chitinase adsorption. While the family 18 chitinases had greater solution activity on chitin oligosaccharides, the family 19 chitinases exhibited greater surface activity on RChitin films, illustrating the importance of CBDs for insoluble substrates.

Improving spinosad production by tuning expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active byproducts in the heterologous host Streptomyces albus J1074.

BACKGROUND: Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-O-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. RESULTS: By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. CONCLUSION: N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus.

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Cloning, expression, homology modelling and molecular dynamics simulation of four domain-containing α-amylase from Streptomyces griseus.

In the present study, α-amylase from Streptomyces griseus TBG19NRA1 was amplified, cloned and successfully expressed in E. coli BL21/DE3. Sequence analysis of S. griseus α-amylase (SGAmy) revealed the presence of four domains (A, B, C and E). Alpha-amylases with E domain (also known as carbohydrate binding module 20 (CBM20)) are capable of degrading raw starch and this property holds great potential for application in starch processing industries. Though α-amylase is a well-studied and characterized enzyme, there is no experimental structure available for this four domain-containing α-amylases. To gain more insight about SGAmy structure and function, homology modelling was performed using a multi-template method. The template α-amylase from Pseudoalteromonas haloplanktis (PDB ID 1AQH) and E domain of Cyclodextrin glucanotransferase from Bacillus circulans (PDB ID 1CGY) was found to have significant similarity with the complete target sequence of SGAmy. Therefore, homology model for SGAmy was generated from the crystal structure of 1AQH and 1CGY and the resulting structure was subjected to 10 ns molecular dynamics (MD) simulation. Remarkably, CBM20 domain of SGAmy showed greater flexibility in MD simulation than other three domains. This observation is highly rational as this part of SGAmy is strongly implicated in substrate (raw starch) binding. Thus, conformational plasticity at CBM20 is functionally beneficial.Communicated by Ramaswamy H. Sarma.

Absorption, translocation, and effects of Bt Cry1Ac peptides from transgenic cotton to the intercrops and soil functional bacteria.

Insecticidal proteins encoded by the truncated genes from Bacillus thuringiensis (Bt) in transgenic crops are released into soil mainly through root exudate and crop residues. In the present study, Bt Cry1Ac protein was hydrolyzed by pronase that was secreted by the soil bacterium Streptomyces griseus. Six peptides were identified as the products of enzymatic hydrolysis by nano liquid chromatography tandem mass spectrometry (LC-MS/MS). One of the six peptides was labeled with radioactive isotope iodine-125 and then purified. The 125I-peptide solution was irrigated to the rhizosphere soil of watermelon seedlings (Citrullus lanatus L.) and wheat seedlings (Triticum aestivum L.), which the two crops usually intercrop with cotton in China. Detection of radioactivity in both plant tissues within one hour proved adsorption, uptake and translocation of the peptide into watermelon and wheat seedlings. Three of the identified peptides were sprayed onto the seedling leaves of watermelon, wheat and maize (Zea mays L.) in the field or the growth chamber. No significant effects on plant growth were observed. These peptides also did not affect growth of organic phosphate-dissolving, nitrogen-fixing, and potassium-dissolving bacteria in the culture. This study provides a new view of GMO risk assessment methodology.

Functional analysis of a novel lytic polysaccharide monooxygenase from Streptomyces griseus on cellulose and chitin.

Lytic polysaccharide monooxygenases (LPMOs) are enzymes that degrade polysaccharides with an oxidative mechanism and contributed to the efficiency in biomass degradation by glycoside hydrolases (GHs). In this study, the substrate and reaction specificity of SgLPMO10A that was an auxiliary activity family 10 (AA10) enzyme with a carbohydrate binding module family 2 (CBM2) domain from Streptomyces griseus, was analyzed. This enzyme produced oxidized cello-oligosaccharides from cellulose and boosted cellulose degradation by cellulases. Detailed study of the AA10 and CBM2 domains revealed that the binding ability of SgLPMO10A depended on CBM2 and that only the AA10 domain functions more effectively in the presence of a certain amount of substrates.

Biotransformation of Erythrodiol for New Food Supplements with Anti-Inflammatory Properties.

Erythrodiol, a typical pentacyclic triterpenic diol in olive oil and its byproduct, olive pomace, frequently appears in food additives for the prevention of cardiovascular diseases because of its antioxidation, anti-inflammatory, and antitumor activities. To develop new derivatives of erythrodiol (1), preparative biotransformations were investigated through Streptomyces griseus ATCC 13273, Penicilium griseofulvum CICC 40293, and Bacillus subtilis ATCC 6633, and ten new (1a-1j) and one known metabolites were isolated. Their structures were elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Furthermore, relative to 1, most metabolites exhibited lower toxicity and more potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In particular, the glycosylated metabolite 1k showed a dramatically increased inhibitory effect with an IC50 value of 2.40 µM, which is even lower than that of quercetin. Thus, biotransformation of erythrodiol is a viable strategy for discovering new triterpenes as food supplements with anti-inflammatory properties.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor.

BACKGROUND: Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. RESULTS: The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. CONCLUSIONS: SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

Spatial structure increases the benefits of antibiotic production in Streptomyces.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.

Cloning and identification of the Frigocyclinone biosynthetic gene cluster from Streptomyces griseus strain NTK 97.

Frigocyclinone is a novel antibiotic with antibacterial and anticancer activities. It is produced by both Antarctica-derived Streptomyces griseus NTK 97 and marine sponge-associated Streptomyces sp. M7_15. Here, we first report the biosynthetic gene cluster of frigocyclinone in the S. griseus NTK 97. The frigocyclinone gene cluster spans a DNA region of 33-kb which consists of 30 open reading frames (ORFs), encoding minimal type II polyketide synthase, aromatase and cyclase, redox tailoring enzymes, sugar biosynthesis-related enzymes, C-glycosyltransferase, a resistance protein, and three regulatory proteins. Based on the bioinformatic analysis, a biosynthetic pathway for frigocyclinone was proposed. Second, to verify the cloned gene cluster, CRISPR-Cpf1 mediated gene disruption was conducted. Mutant with the disruption of beta-ketoacyl synthase encoding gene frig20 fully loses the ability of producing frigocyclinone, while inactivating the glycosyltransferase gene frig1 leads to the production of key intermediate of anti-MRSA anthraquinone tetrangomycin.

Genome-wide screening antifungal genes in Streptomyces griseus S4-7, a Fusarium wilt disease suppressive microbial agent.

Streptomyces is a widely studied bacterial genus, particularly with regard to secondary metabolites and antibiotics production. Streptomyces griseus S4-7 was isolated from a strawberry Fusarium wilt disease suppressive soil, and its biological control ability has been well established. However, the antifungal mechanism of strain S4-7 is not yet fully understood at the molecular and biochemical level. Therefore, in this study we created a random mutant library for strain S4-7 with the Tn5 transposon element to investigate antifungal traits on a genome-wide scale. In total 4646 individual mutant strains were created and 13 mutants were selected based on loss of antifungal activity. The knockout genes were identified as electron transfer oxidoreductase (eto),sigma factor-70(sig70) and nrps by Inverse PCR (I-PCR). eto regulates the geranylgeranyl reductase gene, which is involved in terpenoid-quinone biosynthesis, an important factor in cell fitness. In the △eto strain, expression of wbl, a master regulator of the production of secondary metabolites, was significantly reduced. sig70 is responsible for the cell differentiation sensing mechanism in genus Streptomyces. △nrps showed decreased production of hybrid peptide-polyketide siderophores. These results suggest that S. griseus S4-7 may have various antifungal mechanisms, and each mechanism is essential to maximal antifungal activity.

Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands.

Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC50 values less than 1 µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC50 of 0.391 and 0.0757 µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles.

Regio- and enantioselective O-demethylation of tetrahydroprotoberberines by cytochrome P450 enzyme system from Streptomyces griseus ATCC 13273.

Tetrahydroprotoberberines (THPBs), a class of naturally occurring isoquinoline alkaloids, contain substituent methoxyl or hydroxyl groups which play a significant role in the pharmacological properties of these molecules. In this study, we report a biocatalytic strategy for selective O-demethylation of THPBs. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, exhibited markedly regioselective demethylation of nonhydroxyl-THPBs and monohydroxyl-THPBs on the D-ring. A possible binding mode of THPBs with CYP105D1 was investigated by docking analysis, and the results revealed that the D-rings of THPBs were with the minimum distance to the heme iron. Tetrahydropalmatine was used as a model substrate and enantioselective demethylation was demonstrated. (S)-Tetrahydropalmatine was only demethylated at C-10, while (R)-tetrahydropalmatine was first demethylated at C-10 and then subsequently demethylated at C-9. The kcat/Km value for demethylation of (R)-tetrahydropalmatine by CYP105D1 was 3.7 times greater than that for demethylation of (S)-tetrahydropalmatine. Furthermore, selective demethylation of (S)-tetrahydropalmatine by the CYP105D1-based whole-cell system was demonstrated for the highly efficient production of (S)-corydalmine which has distinct pharmacological applications, such as providing relief from bone cancer pain and reducing morphine tolerance. Moreover, a homologous redox partner was identified to enhance the catalytic efficiency of the CYP105D1-based whole-cell system. This is the first enzymatic characterization of a cytochrome P450 that has regio- and enantioselective demethylation activity of THPBs for application purpose. The cytochrome P450 system could be a promising strategy for selective demethylation in the pharmaceutical industry.

Design, Synthesis and Evaluation of Pentacyclic Triterpenoids Similar to Glycyrrhetinic Acid Via Combination of Chemical and Microbial Modification as Glycogen Phosphorylases Inhibitor.

A series of pentacyclic triterpenoids similar to glycyrrhetinic acid were designed and synthesized via the combination of chemical modification and microbial catalysis. All products were screened for the glycogen phosphorylases inhibitory activities in vitro. Within this series of derivatives, compound 5 displayed good inhibitory activities with IC50 value of 27.7 µM, which is better than that of the other derivatives and glycyrrhetinic acid. Structure-activity relationship (SAR) analysis of these inhibitors was also discussed.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, ß-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

Protection of protein from ruminal degradation by alkali-induced oxidation of chlorogenic acid in sunflower meal.

Lactating ruminants require an adequate supply of absorbable amino acids for the synthesis of milk protein from two sources, that is crude protein (CP) synthesized microbially in the rumen and ruminally undegraded CP (RUP) from feed which can both be digested in the small intestine. Several chemical and physical methods have been identified as being effective in increasing the proportion of RUP of total CP of a feedstuff, yet there is a continuing need for developing and establishing methods which protect feed protein from ruminal degradation with acceptable expenditure of labour and other costs. The objective of this study was to identify and quantify effects of and interactions between chlorogenic acid and protein in solvent-extracted sunflower meal (SFM) as induced by alkali treatment. Response surface methodology was employed to investigate the influence of pH, reaction time and drying temperature on the resulting SFM and, subsequently, its protein value for ruminants estimated from laboratory values. For this purpose, alkali-treated SFM was subjected to a fractionation of feed CP according to the Cornell net carbohydrate and protein system as a basis for estimating RUP at different assumed ruminal passage rates (Kp ). To estimate the intestinal digestibility of the treated SFM and its RUP, a three-step enzymatic in vitro procedure was applied. Alkaline treatment of SFM increased RUP values with factors ranging from approximately 3 (Kp =.08/hr) to 12 (Kp =.02/hr). Furthermore, the intestinal digestibility of the alkali-treated SFM was enhanced by approximately 10% compared to untreated SFM. Increasing pH and reaction time led to both increasing RUP values and intestinal digestibility. In conclusion, a targeted alkaline treatment of naturally occurring compounds in feedstuffs might be a promising approach to provide high-RUP feeds for ruminants which, at the same time, have improved intestinal digestibility values.