Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Establishing microbial co‐cultures for 3‐hydroxybenzoic acid biosynthesis on glycerol

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.     

Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

大肠杆菌中外源基因的表达调节

大肠杆菌已经被广泛地应用于表达各种外源基因,但基因的表达受到多种因素的调节,而且不同的外源基因在大肠杆菌中的表达效率也有很大差异。本文从转录水平调节、翻译水平调节、培养条件调节等方面综述了大肠杆菌中外源基因的表达调节,以便认识其规律,有助于使用有效的方法提高外源基因在大肠杆菌中的表达效率。     

Efficient experimental design and micro‐scale medium enhancement of 6‐deoxyerythronolide B production through Escherichia coli

The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro‐scale cultures and well‐defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6‐deoxyerythronolide B (6dEB) through E. coli. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels of the essential deoxyerythronolide B synthase (DEBS) which catalyze 6dEB conversion. As a result, the study (1) demonstrates the feasibility of micro‐scale cultures to study E. coli 6dEB production and effectively model larger‐scale cultures; (2) identifies an enhanced medium which generates over 160 mg L^?1 6dEB (a 22‐fold improvement over current culture media); and (3) provides new insight and understanding related to the heterologous production of 6dEB from E. coli. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The Logic,Experimental Steps,and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling >50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

基因工程半成品中大肠杆菌蛋白残余量测定方法研究—双抗体夹心ELISA法

用大肠杆菌ＤＨ５α菌体蛋白免疫家兔制备抗血清,建立了双抗体夹心ＥＬＩＳＡ方法。经初步测定,该方法的灵敏度为１．５ｎｇ／ｍｌ,与间接ＥＬＩＳＡ法的灵敏度相似,比聚丙烯酰胺凝胶电泳的考马斯亮兰染色方法的灵敏度高将近７００倍,比银染色方法的灵敏度高将近３０倍。在２０～１００００ｎｇ／ｍｌ范围内呈直线关系,直线相关系数为０．９９６。经八次重复测定,显示很好的重复性。该方法可用于测定基因工程产品中ＤＨ５α菌体蛋白的残余量。     

Biosynthesis of chain‐specific alkanes by metabolic engineering in Escherichia coli

Renewable energy is one of the key issues for sustainable development. Compared with alcohols and esters, alkanes—with the highest energy density—are a better liquid fuel. In this study, we focused on medium‐chain alkanes, the main compounds of jet fuels. To control the chain length of alkanes, a chain length specific thioesterase from Umbellularia californica, a fatty acyl‐CoA reductase Acinetobacter sp. M‐1 that prefers lauroyl‐CoA and myristoyl‐CoA, and a decarbonylase from Nostoc punctiforme were engineering into Escherichia coli cells. The combination of genes, which determines the chain length of products, was carefully designed to control the product spectrum. Undecane and tridecane were produced with a concentration of 2.21 ± 0.18 and 1.83 ± 0.12 mg?g^?1, respectively. A total of 4.01 ± 0.43 mg?g^?1 pentadecane was also detected in the final products. The results showed the feasibility to use microorganisms as cell factories for alkane production. The product spectrum revealed that the chosen genes played a key role in the production of chain length specific alkanes.     

Efficient export of prefolded,disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high‐value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide‐containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild‐type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide‐bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:281–290, 2014     

A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm

We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity. Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures. The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca. eightfold higher as compared to the expression level at 30 degrees C. Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates. Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo. Intact E. coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively. Additionally, the periplasmic fractions were isolated from the three E. coli strains expressing the respective subtilisins and tested for amidase activity. The amidase activity of the three subtilisins was ca. 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions. The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities.     

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His‐tag purification

His‐tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His‐tag is the co‐purification of contaminating histidine‐rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (lo w b ackground str ain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low‐expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His‐tag purifications. Proteins 2013; 81:1857–1861. © 2013 Wiley Periodicals, Inc.     

Expression of key glycosphingolipid biosynthesis‐globo series pathway genes in Escherichia coli F18‐resistant and Escherichia coli F18‐sensitive piglets

A pioneering study showed that the glycosphingolipid biosynthesis‐globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1 and NAGA) may play an important regulatory role in resistance to Escherichia coli F18 in piglets. Therefore, we analysed differential gene expression in 11 tissues of two populations of piglets sensitive and resistant respectively to E. coli F18 and the correlation of differential gene expression in duodenal and jejunal tissues. We found that the mRNA expression of the seven genes was relatively high in spleen, liver, lung, kidney, stomach and intestinal tract; the levels in thymus and lymph nodes were lower, with the lowest levels in heart and muscle. FUT2 gene expression in the duodenum and jejunum of the resistant population was significantly lower than that in the sensitive group (P < 0.01). ST3GAL1 gene expression was also significantly lower in the duodenum of the resistant population than in the sensitive group (P < 0.05). No significant differences were observed among the remaining genes. The expression level of FUT1 was extremely significantly positively correlated with FUT2 and B3GALNT1 expression (P < 0.01) and also had a significant positive correlation with NAGA expression (P < 0.05). The expression level of FUT2 had extremely significant positive correlations with FUT1, ST3GAL1 and B3GALNT1 (P < 0.01). These results suggest that FUT2 plays an important role in E. coli F18 resistance in piglets. FUT1, ST3GAL1, B3GALNT1 and NAGA may also participate in the mechanism of resistance to E. coli F18.