大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。     

聚酮类化合物生物合成的代谢工程研究进展

聚酮化合物是一类重要的具有生物活性的次级代谢物。本文讨论了以聚酮生物合成酶为核心的聚酮化合物生物合成途径,以及近年来有关代谢工程在聚酮类化合物生物合成方面的研究工作进展,主要包括将聚酮生物合成途径引入新的宿主、代谢流量分析在提高聚酮化合物中的应用及合成新的聚酮化舍物等。     

透明质酸的生物合成及其代谢工程的研究进展

透明质酸(hyaluronic acid,HA)是广泛存在于生物体内的功能性糖胺高分子聚合物,在日化、医疗和食品领域应用前景广阔.随着基因工程与代谢工程等合成生物学技术的发展,人们对HA的生物合成过程和机理解析越发深入的同时,也伴随一些新的挑战来临.该综述从分子生物学角度总结了HA的关键合成酶基因及合成途径,对不同来源...     

Metabolic Engineering of Rational Screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L⁻¹ and 129 mg L⁻¹, which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L⁻¹ and 27 mg L⁻¹), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L⁻¹ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.     

代谢工程在芳香化合物生物合成研究中的应用

生物技术和代谢工程的发展促进了生物合成研究。概述了近年来利用微生物莽草酸途径进行芳香化合物生物合成研究的现况、代谢工程在提高天然芳香化合物产量和扩大合成非天然产生的芳香化合物范围的应用的进展 ,特别是整体代谢工程对提高第二代工程菌产量的作用。指出了生物合成法是生产氨基酸及其它生物小分子如奎尼酸、维生素和抗生素等的未来趋势 ,在工业化生产中有着广阔的应用前景。     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

阿维菌素的生物合成及代谢工程研究进展

阿维菌素(avermectin)是由除虫链霉菌(Streptomycesavermitilis)产生的一种具有杀虫活性的大环内酯类抗生素,在农业和畜牧业中应用广泛。本文综述了有关除虫链霉菌基因组序列分析、阿维菌素的生物合成以及阿维菌素育种和代谢工程的研究进展。     

Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.     

代谢工程方法改造大肠杆菌生产胸苷

胸苷是抗艾滋病药物司他夫定(3′-脱氧-2′,3′-双脱氢胸苷)和叠氮胸苷的重要前体物质。应用代谢工程方法对大肠杆菌Escherichia coli BL21(DE3)生物合成胸苷进行了研究。通过敲除E.coli BL21嘧啶回补途径的deo A、tdk和udp三个基因,BS03工程菌株能够积累21.6 mg/L胸苷。为了增加合成胸苷前体物核糖-5-磷酸和NADPH的供给,进一步敲除pgi和pyr L使工程菌BS05胸苷的产量提高到90.5 mg/L。而通过过表达胸苷合成途径的ush A、thy A、dut、ndk、nrd A和nrd B六个基因,菌株BS08胸苷的产量能达到272 mg/L。通过分批补料发酵,BS08最终可以积累1 248.8 mg/L的胸苷。本研究结果表明经过代谢工程改造的E.coli BL21具有良好的胸苷合成能力和应用潜力。     

ubiI,a New Gene in Escherichia coli Coenzyme Q Biosynthesis,Is Involved in Aerobic C5-hydroxylation

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.     

Three-stage assembly of the cysteine synthase complex from Escherichia coli

Control of sulfur metabolism in plants and bacteria is linked, in significant measure, to the behavior of the cysteine synthase complex (CSC). The complex is comprised of the two enzymes that catalyze the final steps in cysteine biosynthesis: serine O-acetyltransferase (SAT, EC 2.3.1.30), which produces O-acetyl-L-serine, and O-acetyl-L-serine sulfhydrylase (OASS, EC 2.5.1.47), which converts it to cysteine. SAT (a dimer of homotrimers) binds a maximum of two molecules of OASS (a dimer) in an interaction believed to involve docking of the C terminus from a protomer in an SAT trimer into an OASS active site. This interaction inactivates OASS catalysis and prevents further binding to the trimer; thus, the system exhibits a contact-induced inactivation of half of each biomolecule. To better understand the dynamics and energetics that underlie formation of the CSC, the interactions of OASS and SAT from Escherichia coli were studied at equilibrium and in the pre-steady state. Using an experimental strategy that initiates dissociation of the CSC at different points in the CSC-forming reaction, three stable forms of the complex were identified. Comparison of the binding behaviors of SAT and its C-terminal peptide supports a mechanism in which SAT interacts with OASS in a non-allosteric interaction involving its C terminus. This early docking event appears to fasten the proteins in close proximity and thus prepares the system to engage in a series of subsequent, energetically favorable isomerizations that inactivate OASS and produce the fully isomerized CSC.     

Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli

Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L ‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034. © 2013 Wiley Periodicals, Inc.