Microbial production of chiral pantothenonitrile through stereospecific reduction of 2′-ketopantothenonitrile

Summary A novel enzymatic process for the synthesis of chiral pantothenonitrile through the asymmetric reduction of 2-ketopantothenonitrile is described.Sporidiobolus salmonicolor AKU 4440 was found to convert 2-ketopantothenonitrile (50 mg/ml) to D-(+)-pantothenonitrile (93.6% e.e.), with a molar yield of 95.6%. In a similar manner, conversion to L-(–)-pantothenonitrile occurred on incubation with washed cells ofRhodotorula glutinis AKU 4845 as the catalyst.     

Microbial conversion of o-acetyltoluidide into 4′-hydroxy-o-acetyltoluidide

Summary Approximately 600 strains of various bacteria and actinomycetes were tested for their ability to hydroxylate o-acetyltoluidide (AT) at the 4'-position. Nocardia asteroides IFO 3384 and N. globerula IFO 13510 were selected as the best strains to catalyse the hydroxylation of AT. The product was isolated from culture] broth and identified as 4'-hydroxy-o-acetyltoluidide (4'-HAT). When AT was added during the course of cultivation of N. asteroides IFO 3384 and N. globerula IFO 13510, final concentrations of 1.0 mg/ml (a 23% molar conversion yield) and 1.5 mg/ml 4'-HAT (a 34% molar conversion yield) were obtained, respectively, from AT after 7 days cultivation. On the other hand, when resting cells of N. globerula IFO 13510 were used, the concentration of 4'-HAT attained was 4.4 mg/ml, a 69% molar conversion yield, after 4 days reaction. Offprint requests to: T. Nagasawa     

Microbial metabolism of monoterpenes — recent developments

Monoterpenes are important renewable resources for the perfume and flavour industry but the pathways and enzymology of their degradation by microorganisms are not well documented. Until recently the acyclic monoterpene alcohols, (+)-camphor and the isomers of limonene were the only compounds for which significant sections of catabolic pathways and associated enzymology had been reported. In this paper recent developments in our understanding of the enzymology of ring cleavage by microorganisms capable of growth with 1,8-cineole and -pinene are described. 1,8-Cineole has the carbocyclic skeleton of a monocyclic monoterpene with the added complication of an internal ether linkage. Ring hydroxylation strategy and biological Baeyer-Villiger oxygenation lead to an efficient method for cleaving the ether linkage. -Pinene is an unsaturated bicyclic monoterpene hydrocarbon. At least two catabolic pathways exist. Information concerning one of them, in which -pinene may be initially converted into limonene, is rudimentary. The other involves attack at the double bond resulting in formation of -pinene epoxide. Ring cleavage is then catalysed by a novel lyase that requires no additional components and breaks both carbocyclic rings in a concerted manner.     

Microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.     

Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose

Guanosine 5’-diphosphate (GDP)-l-fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP-l-fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP-l-fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP-d-mannose, a precursor of GDP-l-fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP-l-fucose concentration (52.5 ± 0.8 mg l^?1) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP-l-fucose concentration of 170.3 ± 2.3 mg l^?1, corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB, manC, gmd and wcaG genes. Further improvement of GDP-l-fucose production was not obtained by additional overexpression of the manA gene.     

Occurrence and metabolism of 1′,4′-trans-diol of abscisic acid

The 1′,4′-trans-diol of abscisic acid was first identified in higher plants with GC-ECD and GC-SIM. The ²H-labelled derivative was converted into abscisic acid (ABA) in pea and avocado, but ²H-labelled ABA was not converted into the diol. These results suggest that the diol is one of the precursors of ABA in higher plants.     

Engineering substrate and energy metabolism for living cell production of cytidine-5′-diphosphocholine

Cytidine-5′-diphosphocholine (CDP-choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP-choline is synthesized by a two-step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one-step living cell fermentation method for CDP-choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP-choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP-choline. Furthermore, co-overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP-choline titer by 55-folds comparing with the wild-type (WT). Overexpression of cytidine-5′-monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP-choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP-choline titer (6.0 g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP-choline bioproduction by living cells.     

Microbial metabolism of α-mangostin isolated from Garcinia mangostana L

α-Mangostin (1), a prenylated xanthone isolated from the fruit hull of Garcinia mangostana L., was individually metabolized by two fungi, Colletotrichum gloeosporioides (EYL131) and Neosartorya spathulata (EYR042), repectively. Incubation of 1 with C. gloeosporioides (EYL131) gave four metabolites which were identified as mangostin 3-sulfate (2), mangostanin 6-sulfate (3), 17,18-dihydroxymangostanin 6-sulfate (4)and isomangostanin 3-sulfate (5). Compound 2 was also formed by incubation with N. spathulata (EYR042). The structures of the isolated compounds were elucidated by spectroscopic data analysis. Of the isolated metabolites, 2 exhibited significant anti-mycobacterial activity against Mycobacterium tuberculosis.     

Microbial production and chemical transformation of poly-γ-glutamate

Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Effective production of retinal from β-carotene using recombinant mouse β-carotene 15,15′-monooxygenase

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg⁻¹. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l⁻¹ β-carotene, 5% (w/v) Tween 40, and 0.2 U ml⁻¹ enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l⁻¹ retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.     

A novel process of inosine 5′-monophosphate production using overexpressed guanosine/inosine kinase

A novel process for producing inosine 5′-monophosphate (5′-IMP) has been demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-l jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5′-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5′-IMP. Received: 7 April 1997 / Received last revision: 18 July 1997 / Accepted: 27 July 1997     

Efficient production of deoxynucleoside-5′-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system

Deoxynucleoside-5′-monophosphates (5′-dNMPs) are the basic components of DNA and are widely used in medicine and as chemical and biochemical reagents. A large amount of effort has been expended to obtain 5′-dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5′-monophosphate (5′-dAMP), 2,6-diaminopurine deoxynucleoside-5′-monophosphate (5′-dDAMP), and deoxycytidine-5′-monophosphate (5′-dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), deoxycytidine kinase, and acetate kinase in a one-pot reaction system. The ndt-II gene from Lactobacillus delbrueckii, dck from Bacillus subtilus, and ack from Escherichia coli K12 were overexpressed in E. coli BL21 (DE3). Thymidine was used as the deoxyribose donor; GTP was used as the phosphate donor, and acetyl phosphate was used to regenerate GTP. Under optimized conditions, each 10 mM adenine, 10 mM 2,6-diaminopurine, or 10 mM cytosine were converted into 9.01 mM 5′-dAMP, 8.68 mM 5′-dDAMP, or 6.23 mM 5′-dCMP, respectively. The high yield indicated that this process of biosynthesis of 5′-dAMP, 5′-dDAMP, or 5′-dCMP was efficient and economical, and this one-pot system may also potentially be used for the preparation of other types of 5′-dNMPs.     

Microbial growth and production kinetics of Streptomyces antibioticus Tü 6040

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1–2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l.     

2′,4′-BNA/LNA aptamers: CE-SELEX using a DNA-based library of full-length 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides

DNA-based aptamers that contain 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) over the entire length were successfully obtained using a capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) method. A modified DNA library was prepared with an enzyme mix of KOD Dash and KOD mutant DNA polymerases. Forty 2′-O,4′-C-methylene bridged/locked nucleic acid (2′,4′-BNA/LNA) aptamers were isolated from an enriched pool and classified into six groups according to their sequence. 2′,4′-BNA/LNA aptamers of groups V and VI bound human thrombin with K_d values in the range of several 10 nanomolar levels.     

Microbial metabolism of quorum-sensing molecules acyl-homoserine lactones, γ-heptalactone and other lactones

The cell-to-cell communication of microorganisms is known to be via exertion of certain chemical compounds (signal molecules) and is referred to as quorum sensing (QS). QS phenomenon is widespread in microbial communities. Several Gram-positive and Gram-negative bacteria and fungi use lactone-containing compounds (e.g. acyl-homoserine lactones (AHLs), γ-heptalactone, butyrolactone-I) as signalling molecules. The ability of microorganisms to metabolise these compounds and the mechanisms they employ for this purpose are not clearly understood. Many studies, however, have focused on identifying AHL and other lactone-degrading enzymes produced by bacteria and fungi. Various strains that are able to utilise these signalling molecules as carbon and energy sources have also been isolated. In addition, several reports have provided evidence on the involvement of lactones and lactone-degrading enzymes in numerous biological functions. These studies, although focused on processes other than metabolism of lactone signalling molecules, still provide insights into further understanding of the mechanisms employed by various microorganisms to metabolise the QS compounds. In this review, we consider conceivable microbial strategies to metabolise AHL and other lactone-containing signalling molecules such as γ-heptalactones.     

Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus

The 5′-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5′ stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses α-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the α-amylase mRNA was proved by measuring α-amylase activity in the culture supernatant and the relative expression of α-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving α-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.