Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g^–1 dry wt.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Improved production of adipate with Escherichia coli by reversal of β-oxidation

The linear C₆ dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

     

Antibiotic resistance and detection of β-lactamase in bacterial strains of Staphylococci and Escherichia coli isolated from foodstuffs

Seventy strains of Staphylococcus spp. and Escherichia coli (35 each) were isolated from various foodstuffs and identified on the basis of cultural, morphological and biochemical characteristics and were further tested for their antibiotic susceptibility with commonly used antibiotics/drugs. 69.2% of the strains of Staphylococci were resistant to co-trimazine and 34.6% were resistant to penicillin-G. 19.2% of the staphylococcal isolates exhibited resistance to cloxacillin, nalidixic acid, methicillin and tetracycline whereas 15.3% of the staphylococcal isolates were resistant to amoxycillin and nitrofurantoin. The isolated E. coli strains exhibited sharp peaks of resistance to antimicrobial agents such as tetracycline (72%), doxycycline (60%) and nalidixic acid (48%). Forty-four percent of the E. coli strains were resistant to nitrofurantoin and penicillin-G respectively. Among the 13 antibiotics/drugs tested for resistance, six different resistance patterns were observed in staphylococcal isolates and seven different resistance patterns were observed in the E. coli isolates from various foodstuffs. Bacterial strains exhibiting MIC values 100 g/ml for ampicillin and cloxacillin were screened for -lactamase activity and out of 10 staphylococcal isolates, seven were found to be positive for -lactamase, whereas out of 13 E. coli isolates tested for -lactamase production, only three were found to be positive.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Kinetics of β-glucosidase production by Escherichia coli recombinants harboring heterologous bgl genes

Maximum productivities of -glucosidase by E. coli recombinants, under the control of either lacZ or GALI promoters, were 33 ± 10 and 100 ± 5 IU l^–1 h^–1, respectively.GAL1 promoter of pYES2 enabled the E. colirecombinants to produce 3.1- and 15.1-fold more -glucosidase than that supported by lacZ promoter of pUC18 in E. coli recombinants and donor, respectively.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Increase in the production of β-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.     

Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement

Fucosyllactoses, including 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2′-FL and 3-FL in engineered Escherichia coli. To boost the production of 2′-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous α1,2-fucosyltransferase and α1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2′-FL and 3-FL. Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2′-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2′-FL and 3-FL biosynthesizing strains. The final concentrations of 2′-FL and 3-FL were 9.12 and 12.43 g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Production of γ-aminobutyric acid in Escherichia coli by engineering MSG pathway

Abstract

The compound γ-aminobutyric acid (GABA) has many important physiological functions. The effect of glutamate decarboxylases and the glutamate/GABA antiporter on GABA production was investigated in Escherichia coli. Three genes, gadA, gadB, and gadC were cloned and ligated alone or in combination into the plasmid pET32a. The constructed plasmids were transformed into Escherichia coli BL21(DE3). Three strains, E. coli BL21(DE3)/pET32a-gadA, E. coli BL21(DE3)/pET32a-gadAB and E. coli BL21(DE3)/pET32a-gadABC were selected and identified. The respective titers of GABA from the three strains grown in shake flasks were 1.25, 2.31, and 3.98?g/L. The optimal titer of the substrate and the optimal pH for GABA production were 40?g/L and 4.2, respectively. The highest titer of GABA was 23.6?g/L at 36?h in batch fermentation and was 31.3?g/L at 57?h in fed-batch fermentation. This study lays a foundation for the development and use of GABA.     

Improved production of lycopene and β-carotene by Blakeslea trispora with oxygen-vectors

Lycopene and β-carotene production were increased when oxygen-vectors, n-hexane and n-dodecane, were added to cultures of Blakeslea trispora because of the enhanced dissolved oxygen concentrations. With 1% (v/v) n-hexane or n-dodecane added in the medium, lycopene production was 51% or 78% higher and β-carotene production was 44% or 65% higher than that of the control, respectively. The highest lycopene and β-carotene production, 533 mg l⁻¹and 596 mg l⁻¹, were obtained when 1% (v/v) n-dodecane and 0.1% (w/v) Span 20 were added together, which were 2.1-fold and 1.8-fold of the control, respectively.     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three -galactosidases (-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing -galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. -Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for -gal I and -gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998