Diacetyl and acetoin production from whey permeate using engineered <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.     

Genome-scale reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of the <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824 metabolic network

To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals, including butanol. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. L. and H. Y. equally contributed to this work.     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Enhancement of daptomycin production in <Emphasis Type="Italic">Streptomyces roseosporus</Emphasis> LC-51 by manipulation of cofactors concentration in the fermentation culture

The effects of eight cofactors of enzymes on daptomycin production were investigated in this work, which included nicotinic acid (VPP), riboflavin (VB₂), heme, thiamine (VB₁), biotin (VH), cyanocobalamin (VB₁₂), tetrahydrofolic acid (THF) and pyridoxal 5-phosphate (VB₆). The dry cell weight (DCW), consumption of glucose, and daptomycin production were obviously improved when proper amount of exogenous cofactors were supplemented in the medium. The effects of heme, THF, VB₁₂ and VB₆ on daptomycin production were especially notable. The daptomycin yield enhanced 363, 104, 53 and 46%, respectively, when optimized amount of these four cofactors were supplemented in the broth. Moreover, the daptomycin yield further increased to 632 mg/l, which was over 4.5-fold higher than that of the control (without cofactors), at 132 h in a 7.5-l fermenter, by supplementation all of the eight cofactors at optimized concentrations (VPP 4 mg/l, VB₂ 0.5 mg/l, heme 9 mg/l, VB₁ 0.4 mg/l, VH 0.1 mg/l, VB₁₂ 0.04 mg/l, THF 6 mg/l and VB₆ 0.4 mg/l). Further, the effects of cofactors on the corresponding key enzymes and important intracellular metabolites were studied in order to elucidate the mechanism of enhancement of daptomycin production by manipulation of cofactors concentration in the fermentation culture. It is suggested that this strategy for increasing the daptomycin production in Streptomyces roseosporus LC-51 by manipulation of cofactors concentration in the fermentation culture may provide an alternative approach to enhance the production of metabolites in other Streptomyces.     

Improvement of Cephalosporin C Production by Recombinant DNA Integration in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Cephalosporins are widely used as anti-infectious β-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.     

Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.     

Increased PCP removal by <Emphasis Type="Italic">Amylomyces rouxii</Emphasis> transformants with heterologous <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> peroxidases supplementing their natural degradative pathway

Fungal peroxidases and phenoloxidases are widely used in aromatic toxic compounds degradation. Peroxidases, such as lignin peroxidase and manganese peroxidase, as well as laccases are mainly produced by basidiomycetes and to a lower extent by other fungi, such as ascomycetes. Peroxidase-encoding genes have been described and homologous expression has been achieved in basidiomycetes. Heterologous expression has also been achieved in some non-producing peroxidase ascomycetes, like Penicillium and Aspergillus. In this work, heterologous expression of peroxidase-encoding genes, lignin peroxidase, and manganese peroxidase was achieved in a zygomycete producing only phenoloxidases (Amylomyces rouxii), aimed at coupling two different pathways used in nature for PCP removal in only one microbial strain. The ability of PCP removal was assayed with one of the obtained transformants, resulting in increased activity with respect to the ability of the parental strain cultured free of the inducer tyrosine (95% and 45%, respectively, of the initial PCP (12.5 mg L⁻¹) in 120 h, or 100% and 49%, respectively, of the initial PCP after 144 h of liquid culture).     

Biotransformation of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide,a novel potent 5‐HT7 receptor antagonist with antidepressant‐like and anxiolytic properties: In vitro and in silico approach

The aim of the study was to investigate the metabolism of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide (PZ‐1150), a novel 5‐HT₇ receptor antagonist with antidepressant‐like and anxiolytic properties, by the following three ways: in vitro with microsomes; in vitro employing Cunninghamella echinulata, and in silico using MetaSite. Biotransformation of PZ‐1150 with microsomes resulted in five metabolites, while transformation with C. echinulata afforded two metabolites. In both models, the predominant metabolite occurred due to hydroxylation of benzene ring. In silico data coincide with in vitro experiments, as three MetaSite metabolites matched compounds identified in microsomal samples. In human liver microsomes PZ‐1150 exhibited in vitro half‐life of 64 min, with microsomal intrinsic clearance of 54.1 μL/min/mg and intrinsic clearance of 48.7 mL/min/kg. Therefore, PZ‐1150 is predicted to be a high‐clearance agent. The study demonstrated the applicability of using microsomal model coupled with microbial model to elucidate the metabolic pathways of compounds and comparison with in silico metabolite predictions.     

Construction of stress-induced metabolic pathway from glucose to 1,3-propanediol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive synthesis. The aim of this study was to engineer an Escherichia coli strain that can produce 1,3-propanediol directly from glucose. We successfully constructed a stress-induced metabolic pathway from glucose to 1,3-propanediol in recombinant E. coli by the expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae and dha operon from Klebsiella pneumoniae, respectively. Batch cultivation of the recombinant E. coli showed that 12.1 g/L 1,3-propanediol was accumulated in the culture without using any inducer.     

Expression of novel carotenoid biosynthesis genes from Enterococcus gilvus improves the multistress tolerance of Lactococcus lactis

Aims

To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid‐producing engineered MG1363 strain under stress condition was investigated.

Methods and Results

We cloned carotenoid biosynthesis genes from yellow‐pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid‐producing L. lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C₃₀ carotenoid 4,4′‐diaponeurosporene. After exposure to 32 mmol l^?1 H₂O₂, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml^?1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7‐, 6.8‐, 8.8‐ and 4.4‐fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100.

Conclusions

The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis.

Significance and Impact of the study

First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.     

Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis

A Lactobacillus brevis strain with the ability to synthesize butanol from glucose was constructed by metabolic engineering. The genes crt, bcd, etfB, etfA, and hbd, composing the bcs-operon, and the thl gene encode the enzymes of the lower part of the clostridial butanol pathway (crotonase, butyryl-CoA-dehydrogenase, two subunits of the electron transfer flavoprotein, 3-hydroxybutyryl-CoA dehydrogenase, and thiolase) of Clostridium acetobutylicum. They were cloned into the Gram-positive/Gram-negative shuttle plasmid vector pHYc. The two resulting plasmids pHYc-thl-bcs and pHYc-bcs (respectively, with and without the clostridial thl gene) were transferred to Escherichia coli and L. brevis. The recombinant L. brevis strains were able to synthesize up to 300 mg l⁻¹ or 4.1 mM of butanol on a glucose-containing medium. A L. brevis strain carrying the clostridial bcs-operon has the ability to synthesize butanol with participation of its own thiolase, aldehyde dehydrogenase, and alcohol dehydrogenase. The particular role of the enzymes involved in butanol production and the suitability of L. brevis as an n-butanol producer are discussed.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Shinella oryzae sp. nov., a novel zearalenone-resistant bacterium isolated from rice paddy soil

A novel bacterium, designated Z-25 ^T, was isolated from a rice paddy rhizosphere soil sample from Wuchang County, China. The Z-25 ^T strain is gram-negative, rod-shaped, non-spore-forming, aerobic, motile by unipolar flagella and straw white in color. A phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain Z-25 belongs to the genus Shinella, and the closest members are Shinella zoogloeoides ATCC 19623 ^T with 98.58% similarity, S. kummerowiae CCBAU 25,048 T (98.03%) and S. granuli Ch06 ^T (97.37%). The average nucleotide identity and in silico DNA-DNA hybridization values between strain Z-25 ^T and the closest members were less than 85.29% and 28.70%, respectively. The predominant fatty acids were the sums of features comprising C_18:1 ω7c and/or C_18:1 ω6c (34.62%), C_18:1 ω7c -11-methyl (20.48%), and C_19:0 cyclo ω8c (18.19%). The only respiratory quinone was ubiquinone-10, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Additionally, a genome analysis showed that Z-25 ^T presented potential functional genes related to the degradation of zearalenone (ZEN). An HPLC analysis indicated that Z-25 ^T could remove 74.13% of 10 mg/L ZEN after 144 h at 30 °C. Therefore, based on phenotypic, chemotaxonomic, phylogenetic and genotypic analyses, strain Z-25 ^T represents a novel species in the genus Shinella, for which the name Shinella oryzae sp. nov. is proposed. The type strain is Z-25 ^T (=?GDMCC 1.2424 ^T?=?KCTC 82660 ^T).

     

Identification of tetracycline‐ and erythromycin‐resistant Gram‐positive cocci within the fermenting microflora of an Italian dairy food product

Aims: Microbiological and molecular analysis of antibiotic resistance in Gram‐positive cocci derived from the Italian PDO (Protected Designation of Origin) dairy food product Mozzarella di Bufala Campana. Methods and Results: One hundred and seven coccal colonies were assigned to Enterococcus faecalis, Lactococcus lactis and Streptococcus bovis genera by ARDRA analysis (amplified ribosomal DNA restriction analysis). Among them, 16 Ent. faecalis, 26 L. lactis and 39 Strep. bovis displayed high minimum inhibitory concentration (MIC) values for tetracycline, while 17 L. lactis showed high MIC values for both tetracycline and erythromycin. Strain typing and molecular analysis of the phenotypically resistant isolates demonstrated the presence of the tet(M) gene in the tetracycline‐resistant strains and of tet(S) and erm(B) in the double‐resistant strains. Southern blot analysis revealed plasmid localization of L. lactis tet(M), as well as of the erm(B) and tet(S) genes. Genetic linkage of erm(B) and tet(S) was also demonstrated by PCR amplification. Conjugation experiments demonstrated horizontal transfer to Ent. faecalis strain JH2‐2 only for the plasmid‐borne L. lactis tet(M) gene. Conclusions: We characterized tetracycline‐and erythromycin‐resistance genes in coccal species, representing the fermenting microflora of a typical Italian dairy product. Significance and Impact of the Study: These results are of particular relevance from the food safety viewpoint, especially in the light of the potential risk of horizontal transfer of antibiotic‐resistance genes among foodborne commensal bacteria.     

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.     

Production and genetic improvement of a novel antimycotic agent,Saadamycin, against Dermatophytes and other clinical fungi from Endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. Hedaya48

As a part of our ongoing efforts towards finding novel antimycotic agents from marine microflora of the Red Sea, vanillin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and the new antimycotic compound saadamycin were isolated from endophytic Streptomyces sp. Hedaya48. The producing strain was isolated from the Egyptian sponge Aplysina fistularis and subjected to different UV irradiation doses. A mutant strain Ah22 with 10.5-fold (420 mg/l as compared to 40 mg/l produced by the parental strain) improved saadamycin production was isolated. Production of saadamycin from mutant Ah22 was enhanced to 2.26-fold (950 mg/l) and 2.38-fold (1000 mg/l) under optimized culture conditions in batch culture and bioreactors, respectively. Both saadamycin and 5,7-dimethoxy-4-p-methoxylphenylcoumarin exhibited significant antimycotic activity against dermatophytes and other clinical fungi.