Increase in Silk Production by the Silkworm,Bombyx morì L., due to Oral Administration of a Juvenile Hormone Analog

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3?h^?1) and pH (5.5); the 1,3-BD concentration reached 98.5?mM after 36?h with high-yield (0.444?mol (mol glucose)^?1) and a high maximum production rate (3.63?mM?h^?1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8?mM 1,3-BD after 96?h cultivation with a yield of 0.372?mol (mol glucose)^?1, a maximum production rate of 3.90?mM?h^?1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Metabolic engineering of acetoin and meso-2, 3-butanediol biosynthesis in E. coli

The functional reconstruction of acetoin and meso-2,3-butanediol (meso-2,3-BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vsivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA⁽ ilvC( expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-BD, suggesting the existence of endogenous 2,3-BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3-BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-BD was found to be the most important consequence of ldhA deletion.     

Asymmetric reduction of 4-hydroxy-2-butanone to (R)-1,3-butanediol with absolute stereochemical selectivity by a newly isolated strain of Pichia jadinii

In this study, a novel strain of Pichia jadinii, HBY61, capable of the biocatalysis of 4-hydroxy-2-butanone (4H2B) to (R)-1,3-BD was isolated. HBY61 produced (R)-1,3-BD with high activity and absolute stereochemical selectivity (100 % e.e). Glucose and beef extract were found to be the key factors governing the fermentation, and their optimal concentrations were determined to be 84.2 and 43.7 g/L, respectively. The optimal bioconversion conditions of 4H2B catalyzed by HBY61 were pH 7.4, 30 °C, and 250 rpm with 6 % (v/v) glucose as the co-substrate. Accordingly, when 45 g/L of 4H2B was divided into three equal parts and added successively into the system at set time intervals, the maximum (R)-1,3-BD concentration reached 38.3 g/L with high yield (85.1 %) and strict 100 % enantioselectivity. Compared with previously reported yields for the biocatalytic production of (R)-1,3-BD, the use of strain HBY61 provided a high yield with excellent stereoselectivity.     

Microbial production of 4-hydroxybutyrate,poly-4-hydroxybutyrate,and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms

4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.     

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Metabolism of 3-hydroxybenzoate and gentisate by strain <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP

The ability of strain Rhodococcus opacus 1CP to utilize 3-hydroxybenzoate (3-HBA) and gentisate in concentrations up to 600 and 700 mg/L, respectively, as sole carbon and energy sources in liquid mineral media was demonstrated. Using high-performance liquid chromatography (HPLC) and thin-layer chromatography, 2,5-dihydroxybenzoate (gentisate) was identified as the key intermediate of 3-hydroxybenzoate transformation. In the cell-free extracts of the strain grown on 3-HBA or gentisate, the activities of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, and maleylpyruvate isomerase were detected. During growth on 3-HBA, low activity of catechol 1,2-dioxygenase was detected. Based on the data obtained, the pathway of 3-HBA metabolism by strain R. opacus 1CP was proposed.     

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.     

Stereoselective enzymatic hydrolysis of 2-cyclohexyl-and 2-phenyl-1,3-propanediol diacetate in biphasic systems

Summary A key intermediate (S(–) 2-cyclohexyl-1,3-propanediol monoacetate) was made with high optical purity for the total synthesis of a new angiotensin converting enzyme inhibitor, Fosinopril. The stereoselective hydrolysis of 2-cyclohexyl-1,3-propanediol diacetate (I) and 2-phenyl-1,3-propanediol diacetate (II) was carried out with lipases. Among various lipases evaluated, only porcine pancreatic lipase (PPL) and Chromobacterium viscosum lipase demonstrated efficient conversion and gave the desired enantiomer of monoacetate. In aqueous solution, the desired S(–) monoacetate exhibited an optical purity of 65%–80% (30%–60% enantiomeric excess [e.e.]). However, when the same reactions were conducted in a biphasic system, the product S(–) monoacetate exhibited an optical purity of 99%–100% (98%–100% e.e.). The high purity product was achieved with 65 mol% yield at 1% substrate concentration. Among various solvents evaluated in biphasic systems, efficient hydrolysis was achieved in toluene, cyclohexane, and trichloro-trifluoroethane. The crude PPL was partially purified and two lipase fractions (A and B) were identified. Lipases A and B had a molecular mass of 38 000 and 40 000 daltons, respectively, and both were found to catalyze the hydrolysis of I and II to the appropriate monoacetate in a biphasic system. Offprint requests to: R. N. Patel     

转化奎宁酮为(R)-3-奎宁醇菌株的筛选及转化条件优化

(R)-3-奎宁醇是一种用于合成各类药物的重要手性砌块,以奎宁酮盐酸盐为唯一碳源,筛选得到一株能够将奎宁酮不对称还原为(R)-3-奎宁醇的菌株X15。常规生理生化鉴定和18S rDNA序列分析表明,菌株X15属于粘红酵母菌Rhodotorula mucilaginosa,定名为R.mucilaginosa X15。结果显示,菌株X15具有酮基还原能力和辅酶再生能力,在100 mL反应体系中可将奎宁酮还原为(R)-3-奎宁醇,转化率90%,ee值为88%。     

Production of D-lactic acid from 1,2-propanediol byPseudomonas sp. strain TB-135

Summary A newly isolated bacterium, strain TB-135, produces solely D-lactic acid from 1,2-propanediol (1,2-PD). From taxonomical studies, the strain was concluded to belong to the genusPseudomonas. The optimal conditions for the acid production were found to be at 4% 1,2-PD and 0.2% yeast extract, when the strain produced 21mg/ml of the acid of a high optical purity (over 99% e.e.) after 4days of cultivation. Since it was considered that the acid was produced from (R)-(–)-1,2-PD, the molar yield was estimated to be 86%.     

Microbial production of meso-2,3-butanediol by metabolically engineered Escherichia coli under low oxygen condition

A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.     

Stereoselective microbial reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide

Several microbial cultures were screened for the ability to catalyse the reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (1). The chiral intermediate (+)N-(4-(1-hydroxy-2-chloroethyl) phenyl methane sulfonamide (2) was prepared by the stereoselective microbial reduction of the parent ketone 1. Compound 2 is a potential chiral intermediate for synthesis of 4-(2-isopropylamino-1-hydroxyethyl) phenyl methanesulfonanilide (d-sotalol), a beta-receptor antagonist. Microorganisms from the genera Rhodococcus, Nocardia, and Hansenula reduced 1 to 2. A reaction yield of >50% and optical purities of >90% were obtained. The best strain (H.polymorpha ATCC 26012) effectively reduced compound 1 to compound 2 in 95% reaction yield and 99% optical purity. Compound 2 (8.2 g) was isolated from a 3-1 preparative batch in 68% overall yield. Isolated compound 2 had a specific rotation of +20° (CH₂Cl₂, C-1), an optical purity of 99.5%, and a chemical purity of 97% as analyzed by gas chromatography and HPLC. The nuclear magnetic resonance and mass spectra of compound 2 prepared by bioreduction and a standard chemical sample of 2 were virtually identical. Cell extracts of H. polymorpha in the presence of glucose dehydrogenase, glucose and nicotinamide adenine dinucleotide (NAD ⁺) catalyzed the reduction of 1 to 2 with 98% reaction yield and resulted in an optical purity of 99.4%. Correspondence to: R. N. Patel     

Synthesis of (S)- and (R)-3-hydroxy acids using cells or purified (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum and the NADP(H) regeneration system of Clostridium thermoaceticum

A purified and partially characterized novel NADP⁺-dependent oxidoreductase from Clostridium tyrobutyricum DSM 1460 was applied for the preparative reduction of several 3-oxo acids to (S)-3-hydroxy acids. (R)-3-Hydroxybutyrate was prepared by the same enzyme selectively dehydrogenating the S enantiomer of (R,S)-3-hydroxybutyrate. The enantiomeric purity of the (S)- and (R)-3-hydroxy acids was at least 98% enantiomeric excess (e.e). NADPH for reductions and NADP⁺ for dehydrogenations were regenerated by applying artificial mediator accepting pyridine nucleotide oxidoreductases in the form of a crude extract of C. thermoaceticum cells. For NADP⁺ regeneration also the system 2-oxoglutarate/glutamate dehydrogenase was used for comparison. Instead of the purified (S)-3-hydroxycarboxylate oxidoreductase, resting cells of C. tyrobutyricum were also applied for reductions and dehydrogenations with substrate concentrations of 200–400 mM leading to products with e.e. values above 96%.Dedicated to Prof. H.G. Floss on the occasion of his 60th birthday     

Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate that 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.Abbreviations TNT 2,4,6-trinitrotoluene - 1,3DNB 1,3-dinitrobenzene - 4NC 4-nitrocatechol - 3NA 3-nitroaniline - NB nutrient broth; t_d doubling time - OD₅₄₆ optical density at 546 nm     

Reduction of 2-Substituted Cyclohexanones by Saccharomyces Cerevisiae

An enzymatic reduction of 2-substituted cyclohexanones mediated by Saccharomyces cerevisiae was studied with respect to the stereochemical course and optical purity of the products. Reduction of ketones 1b-1f resulted in separable diastereoisomeric mixtures of cis- and trans-stereoisomers of 2-substituted cyclohexanols (2b-2f and 3b-3f) having the (S) absolute configuration at the chiral center bearing the hydroxyl functionality with high enantiomeric purity. Reduction of ketone 1a yielded mixture of cis-(1S, 2R)- and trans-(1R, 2R)-stereoisomers (2a and 3a) with lower enantiomeric purity. Changes in the nature of the C(2)-substituent affect the stereochemical course of the biotransformation. However, they significantly influenced the enantiomeric purity of the products. The diastereoselectivity of the process was studied as well; high diastereoselectivity was observed with the substrates 1a, 1e and 1f.     

Production of (R)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by Corynebacterium sp. Strain N-1074

The production of (R)-3-chloro-1,2-propanediol [(R)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a Corynebacterium strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (R)-MCP production by the resting cell reaction was around 8.0. The optical purity of (R)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (R)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.     

Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308

A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5?g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30?g/L glucose, respectively), and 25.3?g/L 2,3-BD from molasses (at its initial concentration equivalent to 60?g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25?g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50?g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004