重组大肠杆菌利用D-木糖合成D-1,2,4-丁三醇

1,2,4-丁三醇(1,2,4-butanetriol, BT)是一种重要的有机合成中间体。通过克隆表达恶臭假单胞菌(Pseudomonas putida ATCC12633)2-酮酸脱羧酶(mdlC)和新月柄杆菌(Caulobacter crescentus CB15)D-木糖脱氢酶(xdh),敲除木糖利用和D-1,2,4-丁三醇合成中间代谢物分解途径中关键基因木糖异构酶(xylA)和2-酮酸醛缩酶(yjhH和yagE),重构大肠杆菌代谢网络,得到了能够将D-木糖转化为D-1,2,4-丁三醇的重组菌株。考察了温度、装液量、pH控制等条件对重组菌株合成D-1,2,4-丁三醇的影响,在适宜条件下发酵36 h后D-1,2,4-丁三醇产量达到3.96 g·L^-1。探讨了葡萄糖利用与丁三醇合成的关系,通过敲除编码酶IICB^Glc的ptsG基因改造重组菌株的磷酸烯醇式丙酮酸葡萄糖转移酶(phosphoenolpyruvate: sugar phosphotransferase system, PTS)系统,菌株可以在利用葡萄糖生长的同时进行木糖的转化,具有更高的合成能力。     

大肠杆菌偏利共培养系统合成大豆苷元

大豆苷元是一种植物雌激素,具有多种生物活性,但在大肠杆菌中的生物全合成还未见报道。基于大豆苷元合成途径的三个模块（对香豆酸、甘草素和大豆苷元模块）,构建大肠杆菌共培养系统从头合成大豆苷元。将对香豆酸和甘草素模块分配到两株大肠杆菌中构建双菌共培养系统,合成甘草素。在此基础上,探索了三种共培养模式合成大豆苷元,结果显示,三菌共培养系统比其他两种双菌共培养系统的产量更高,达到27.8 mg/L。共培养菌株间通过苯丙氨酸的单向流动形成了偏利共生的关系,有助于平衡代谢途径,提高大豆苷元产量。该共培养系统在大肠杆菌中实现大豆苷元的从头合成,为其他黄酮类化合物的生物合成提供了即插即用的平台。     

Biosynthesis of 4-hydroxyphenylpyruvic acid from l-tyrosine using recombinant Escherichia coli cells expressing membrane bound l-amino acid deaminase

4-Hydroxyphenylpyruvic acid(4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase(mL-AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H_2 O_2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide(twin-arginine translocation pathway, Tat) and integrated pelB(the general secretory pathway, Sec)-Tat signal peptide was determined and compared firstly, using two pET systems(pET28 a and pET20 b). It was found that the Tat pathway(pET28 a-mlaad) resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts B121(DE3) and 0.05 mmol ·L~(-1) IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol·L~(-1) 4-HPPA was obtained from 100 mmol·L~(-1) tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.     

产高纯度D-乳酸大杨杆菌的构建及发酵

以可利用蔗糖的Escherichia coli W为出发菌株,敲除产生副产物的相关基因(adhE, frdBC, pta, pflB, aldA),为促进蔗糖利用,还敲除蔗糖启动子的抑制基因(cscR),构建了D-乳酸工程菌WD 206. 结果表明,该菌经72 h发酵可有效将100 g/L蔗糖转化生成88.15 g/L乳酸,产率为84%,占代谢产物的99.5%, D-乳酸的光学纯度达99%.     

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.     

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Enzyme engineering has made impressive progress in the past decades, paving the way for the widespread use of enzymes for various purposes. In contrast to “classical” enzyme engineering, which focuses on optimizing specific properties of natural enzymes, a more recent trend towards the creation of artificial enzymes that catalyze fundamentally distinct, new-to-nature reactions is observable. While approaches for creating such enzymes differ significantly, they share the common goal of enabling biocatalytic novelty to broaden the range of applications for enzymes. Although most artificial enzymes reported to date are only moderately active and barely function in vivo, they have the potential to endow cells with capabilities that were previously out of reach and thus herald a new wave of “functional xenobiology”. Herein, we highlight recent developments in the field of artificial enzymes with a particular focus on challenges and opportunities for their use in xenobiology.     

Taking Synthetic Biology to the Seas: From Blue Chassis Organisms to Marine Aquaforming

Oceans cover 71 % of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions, and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example of the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed.     

Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.     

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.     

Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation

Animal-derived protein production is one of the major traditional protein supply methods, which continues to face increasing challenges to satisfy global needs due to population growth, augmented individual protein consumption, and aggravated environmental pollution. Thus, ensuring a sustainable protein source is a considerable challenge. The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins, which is an important way to solve the protein supply problem. This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis. Moreover, using artificial bioengineered milk and artificial bioengineered eggs as examples, the progress of food protein supply transition based on synthetic biology has been systematically summarized. Additionally, the future of food synthetic biology as a potential source of protein has been also discussed. By strengthening and innovating the application of food synthetic biology technologies, including genetic engineering and high-throughput screening methods, the current limitations of artificial foods for protein synthesis and production should be addressed. Therefore, the development and industrial production of new food resources should be explored to ensure safe, high-quality, and sustainable global protein supply.     

Protein interactions in the Escherichia coli cytosol: an impediment to in-cell NMR spectroscopy

Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.     

Ligand- and Structure-Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence

Enoyl-acyl carrier protein reductase (FabI) is the limiting step to complete the elongation cycle in type II fatty acid synthase (FAS) systems and is a relevant target for antibacterial drugs. E. coli FabI has been employed as a model to develop new inhibitors against FAS, especially triclosan and diphenyl ether derivatives. Chemical similarity models (CSM) were used to understand which features were relevant for FabI inhibition. Exhaustive screening of different CSM parameter combinations featured chemical groups, such as the hydroxy group, as relevant to distinguish between active/decoy compounds. Those chemical features can interact with the catalytic Tyr156. Further molecular dynamics simulation of FabI revealed the ionization state as a relevant for ligand stability. Also, our models point the balance between potency and the occupancy of the hydrophobic pocket. This work discusses the strengths and weak points of each technique, highlighting the importance of complementarity among approaches to elucidate EcFabI inhibitor's binding mode and offers insights for future drug discovery.     

Towards Functional Orthogonalisation of Protein Complexes: Individualisation of GroEL Monomers Leads to Distinct Quasihomogeneous Single Rings

The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single‐ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.     

Intensification of chemical separation engineering by nanostructured channels and nanofluidics: From theories to applications

With the development of manufacturing technology on the nanoscale, the precision of nano-devices is rapidly increasing with lower cost. Different from macroscale or microscale fluids, many specific phenomena and advantages are observed in nanofluidics. Devices and process involving and utilizing these phenomena play an important role in many fields in chemical engineering including separation, chemical analysis and transmission.In this article, we summarize the state-of-the-art progress in theoretical studies and manufacturing technologies on nanofluidics. Then we discuss practical applications of nanofluidics in many chemical engineering fields,especially in separation and encountering problems. Finally, we are looking forward to the future of nanofluidics and believe it will be more important in the separation process and the modern chemical industry.     

Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine

L-Serine O-acetyltransferase (SAT) from Escherichia coli catalyzes the first step of L-cysteine synthesis in E.coli and is strictly inhibited by the second step product, L-cysteine. To establish a fermentation process to produce L-cysteine, we embarked on a mutational study of E.coli SAT to desensitize the feedback inhibition by L-cysteine. The crystal structure and the reaction mechanism of SAT from E.coli have shown that the substrate L-serine and the inhibitor L-cysteine bind to the identical region in the SAT protein. To decrease the affinity for only L-cysteine, we first built the structure model of L-serine-binding SAT on the basis of the crystal structure with bound L-cysteine and compared these two structures. The comparison showed that the Calpha of Asp92 underwent a substantial positional change upon the replacement of L-cysteine by L-serine. We then introduced various amino acid substitutions at positions 89-96 around Asp92 by randomized, fragment-directed mutagenesis to change the position of the Asp92. As a result, we successfully obtained mutant SATs which have both extreme insensitivity to an inhibition by L-cysteine (the concentration that inhibits 50% activity; IC(50) = 1,100 micromol/l, the inhibition constant; K(i) = 950.0 micromol/l) and extremely high emzymatic activities.