Xinglin Jiang

Biosynthetic pathways for 3-hydroxypropionic acid production

Biobased platform chemicals have attracted growing interest recently. Among them, 3-hydroxypropionic acid receives significant attention due to its applications in the synthesis of novel polymer materials and other derivatives. To establish a biotechnology route instead of the problematic chemical synthesis of 3-hydroxypropionic acid, biosynthetic pathway is required, and the strategies of how to engineer a microbe to produce this product should be considered. In the present review, we summarize and review all known pathways, which could be potentially constructed for 3-hydroxypropionic acid production. Mass and redox balances are discussed in detail. Thermodynamic favorability is evaluated by standard Gibbs free energy. The assembly of pathways and possible solutions are proposed. Several new techniques and future research needs are also covered.

Enhancing Production of Bio-Isoprene Using Hybrid MVA Pathway and Isoprene Synthase in E. coli

The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the “upper pathway” which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation.

Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene.

Backgroundα-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, α-Pinene used in industry is mainly produced either by tapping trees (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, the extraction of it from trees is tedious and inefficient and requires substantial expenditure of natural resources. Therefore, it is necessary to seek sustainable technologies for α-pinene production.ResultsTo construct the microbial synthetic pathway of α-pinene in E. coli, we co-expressed native geranyl diphosphate synthase (IspA) from E. coli and α-pinene synthase (Pt30) from Pinus taeda, and then to increase the geranyl diphosphate (GPP) content in the cells, a suitable geranyl diphosphate synthase (GPPS2) was selected from two different origins. Furthermore, to enhance α-pinene production, a novel biosynthetic pathway of α-pinene was assembled in E. coli BL21(DE3) with the heterologous hybrid mevalonate (MVA) pathway, GPPS2 and α-pinene synthase (Pt30). The final genetic strain, YJM28, harboring the above novel biosynthetic pathway of α-pinene, accumulated α-pinene up to 5.44 mg/L and 0.97 g/L under flask and fed-batch fermentation conditions, respectively. The conversion efficiency of glucose to α-pinene (gram to gram) in the metabolically engineered strain reached 2.61%.ConclusionsIn this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of α-pinene was successfully assembled in E. coli BL21(DE3) with the heterologous hybrid MVA pathway, GPPS2 and α-pinene synthase (Pt30). In addition, this is the first report on α-pinene fed-batch fermentation, and our results represent improvements over previous reports.

Bio-isoprene production using exogenous MVA pathway and isoprene synthase in Escherichia coli

In this paper, an original strategy is employed to biosynthesize the isoprene by heterologously co-expressing the Saccharomyces cerevisiae MVA pathway and isoprene synthase (IspS) from Populus alba in the Escherichia coli BL21 (DE3) strain, which was screened from three different IspS enzymes. The finally genetic strain YJM13 harboring the MVA pathway and ispS(Pa) gene could accumulate isoprene up to 2.48 mg/l and 532 mg/l under the flask and fed-batch fermentation conditions, respectively, which is about three times and five times to the control strain. The result proves to be higher than that in the report documents. In this way, a potential production system for isoprene from renewable sources via the MVA pathway in E. coli has been provided.

Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

BackgroundThioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.ResultsWe cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.ConclusionsFor the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.

Recovery Processes of Organic Acids from Fermentation Broths in the Biomass-Based Industry

The new movement towards green chemistry and renewable feedstocks makes microbial production of chemicals more competitive. Among the numerous chemicals, organic acids are more attractive targets for process development efforts in the renewable-based biorefinery industry. However, most of the production costs in microbial processes are higher than that in chemical processes, among which over 60% are generated by separation processes. Therefore, the research of separation and purification processes is important for a promising biorefinery industry. This review highlights the progress of recovery processes in the separation and purification of organic acids, including their advantages and disadvantages, current situation, and future prospects in terms of recovery yields and industrial application.

In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

Biodegradation-inspired bioproduction of methylacetoin and 2-methyl-2,3-butanediol

Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l−1 for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.

Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties

ABSTRACT Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.

A New Nitrilase Producing Pantoea sp. and Its Enzymological Properties: A New Nitrilase Producing Pantoea sp. and Its Enzymological Properties

Nitrilases can hydrolyze nitrile efficiently under mild conditions. For their low pollution and cost, the enzymatic methods with nitrilases are widely used in agriculture, industry, biomedicine and environment protection. Nitrilases with broad substrate specificity are lacked in the nitrilase market. In this study, Berthelot method and high performance liquid chromatography were used to separate and identify a new strain of nitrilase Pantoea sp. 1-2, which can hydrolyze acetonitrile, 4-hydroxyphenylcyanide and 3-hydroxypropionitrile. The strain produced 16.45 U/mL nitrilase with ammonium sulfate as nitrogen source. The enzyme remained 80% activity at pH 6.6-7.6, temperature 22-45 °C, staying stable after 30 h incubation. The strain would be of great value in industrial utilization. Fig 7, Ref 21