Laboratory-scale production of (S)-reticuline,an important intermediate of benzylisoquinoline alkaloids,using a bacterial-based method

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.     

Process and metabolic strategies for improved production of Escherichia coli-derived 6-deoxyerythronolide B

Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced approximately 100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.     

Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity

Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.     

海洋中海藻糖产生菌的筛选及发酵条件优化

从180余份海水、海泥样品中筛选得到60株产海藻糖较高的菌株,编号为2-14的菌株海藻糖产量最高,为127.9mg/g cell。对2-14菌株进行形态特征、培养特征及生理生化试验,鉴定该菌株为红酵母属(Rhodotorula sp.)。研究摇瓶发酵条件对红酵母海藻糖产量的影响,结果为:初始pH5.5,发酵温度28℃,装液量75mL(250mL三角瓶中)。采用优化后发酵条件红酵母海藻糖产量为193.3mg/g cell,优化前对照值为132.1mg/g cell,优化后的结果是优化前的1.46倍。在5L发酵罐中培养得到最佳发酵时间为54h,发酵罐培养发酵液中海藻糖含量最高达2.5g/L,为摇瓶培养的1.6倍。     

Medium optimization of transformed root cultures of Stizolobium hassjoo producing L-DOPA with response surface methodology

Medium optimization of B5 medium for hairy root cultures producing secondary metabolites was studied through statistical experimental design. Transformed root cultures of Stizolobium hassjoo producing L-DOPA were used as a model system. The serial dilution experiments facilitated logical choice of the upper and lower bounds on executing 2(11)(-)(6) fractional factorial design. Steepest ascent method as well as central composite design were sequentially employed to optimize the media of shake flask cultures. The modified B5 media of GM, PM, and GPM were obtained, indicating the optimum medium compositions for enhancing hairy root dry weight, L-DOPA content in hairy roots, and L-DOPA production, respectively. When cultivating S. hassjoo hairy roots in GM, PM and GPM for 16 days, the dry wt of hairy roots, L-DOPA content, and L-DOPA production obtained were ca. 530 mg per flask (10.6 g/L), 10.8% dry wt, and 806 mg/L, which were 1.8-, 2-, and 2.8-fold of basal B5 medium control runs, respectively.     

Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10(T) and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10(T) and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

纤维素硫酸钠-聚二甲基二烯丙基氯化铵微胶囊固定化Candida krusei产甘油条件优化

研究了利用一种新型的纤维素硫酸钠／聚二甲基二烯丙基氯化铵（Nacs／PDMDAAc）中空微胶囊包埋Can—dida krusei ZJU5205制备甘油的过程。通过摇瓶培养过程中对初始包菌量、胶囊和胶珠、初始甘油浓度、胶囊大小、胶囊体积／发酵液体积等关键固定化参数和培养条件的优化,确立了NaCS／PDMDAAC微胶囊固定化Candida krusei的最佳工艺参数为包菌量为0．6g／L,发酵初加入20g／L甘油,胶囊体积／发酵液体积为0．4。     

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration.     

Production of recombinant bacteriocin divercin V41 by high cell density <Emphasis Type="Italic">Escherichia coli</Emphasis> batch and fed-batch cultures

To increase the yield of heterologous production of the class II bacteriocin DvnRV41 with Escherichia coli Origami (DE3) (pLysS/pCR03), induction of bacteriocin gene expression was optimized by varying the inducer isopropyl beta-D-thiogalactopyranoside (IPTG) concentration (0-2 mM), and controlled batch and fed-batch cultures were tested on a 2-L scale. A concentration of 0.5 mM IPTG was found to be optimal for cell growth and bacteriocin production. Shake flask cultivation of E. coli Origami (DE3) (pLysS/pCR03) gave biomass and bacteriocin yields of 1.54 +/- 0.06 g cdw/l and 18 +/- 1 mg DvnRV41/l, respectively. Biomass (2.70 +/- 0.06 and 6.8 +/- 0.6 g cdw/l, respectively) and bacteriocin yields (30 and 74 mg DvnRV41 per liter, respectively) were both increased with batch and fed-batch compared to shake flask cultures. Bacteriocin yields reported in this study are among the highest published for other heterologous expression systems in shake flasks.     

Synthesis and excretion of alpha-amylase in vgb+ and vgb- recombinant Escherichia coli: a comparative study

Synthesis and excretion of alpha-amylase is investigated in batch cultures of Escherichia coli JM103[pMK57] (vgb-) and E. coli JM103[pMK79] (vgb+). While total production and excretion of alpha-amylase were promoted in Luria broth (LB) (excretion being as high as 87%), cell-mass-specific production of the enzyme was promoted in M9 in bioreactor cultures and in LB in shake flask cultures. Low aeration and agitation rates and presence of starch were conducive to alpha-amylase synthesis in E. coli JM103[pMK79]. Two-stage bioreactor operating strategies that will improve alpha-amylase production are proposed. The potential of these strategies is demonstrated via two-stage shake flask cultures.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies

The expression of IgG antibodies in Escherichia coli is of increasing interest for analytical and therapeutic applications. In this work, we describe a comprehensive and systematic approach to the development of a dicistronic expression system for enhanced IgG expression in E. coli encompassing: (i) random mutagenesis and high-throughput screening for the isolation of over-expressing strains using flow cytometry and (ii) optimization of translation initiation via the screening of libraries of synonymous codons in the 5' region of the second cistron (heavy chain). The effects of different promoters and co-expression of molecular chaperones on full-length IgG production were also investigated. The optimized system resulted in reliable expression of fully assembled IgG at yields between 1 and 4 mg/L of shake flask culture for different antibodies.     

重组毕赤酵母生产β-葡萄糖苷酶发酵条件初步研究

为提高重组毕赤酵母(P.pastoris KM71/pPIC9K-bgl)生产β-葡萄糖苷酶的产量,在摇瓶条件下对重组P.pastoris产β-葡萄糖苷酶的发酵过程进行了优化,得到最佳的条件:生长阶段甘油浓度为30 g/L,接种量为10%,诱导阶段甲醇的初浓度为4%,过程补加甲醇0.5%,诱导温度30℃,pH7.5,诱导周期120 h,酶活可达到245 U/mL。在此基础上,在3 L发酵罐上进行初步放大,流加甘油提高细胞密度至OD_(600)为170,开始流加甲醇诱导,最终BGL酶活达到1 175 U/mL。比摇瓶提高了4.8倍,为β-葡萄糖苷酶工业化生产打下了坚实的基础。     

An automated home-built low-cost fermenter suitable for large-scale bacterial expression of proteins in Escherichia coli

We have developed an automated fermentation system for cost-efficient upscaling of protein expression in bacteria. The system, built for use by nonbiotechnologists, can be assembled mostly from standard laboratory equipment and allows a largely unattended growth of bacteria to OD 25 (at 600 nm) in a 12 L vessel. The typical yield of 250-350 g of wet weight cell pellet per run, which is equivalent to the biomass obtained from 250 shake flask cultures containing 400 mL Luria-Broth medium each, facilitates the production of large amounts of purified recombinant protein without the laborious need for optimization of expression and purification conditions.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products.