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Item type:Publication, Access status: with files , Strain evolution and novel downstream processing with integrated catalysis enable highly efficient coproduction of 1,3-propanediol and organic acid esters from crude glycerolBioconversion of natural microorganisms generally results in a mixture of various compounds. Downstream processing (DSP) which only targets a single product often lacks economic competitiveness due to incomplete use of raw material and high cost of waste treatment for by-products. Here, we show with the efficient microbial conversion of crude glycerol by an artificially evolved strain and how a catalytic conversion strategy can improve the total products yield and process economy of the DSP. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72, and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The coproduction of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for the simultaneous production of three appealing products from the crude glycerol fermentation broth, which greatly improve the process economy and ecology.Publicationtype: Journal ArticleScopus© Citations 9 71 209 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Analysis of co-evolved interaction network of E.coli aspartokinase III and regulation of allosteric progress using energy transduction(2022-02-01); ; Understanding co-evolved interaction network involved in allosteric regulation of kinase is of fundamental interest. Here, with aspartokinase III (AKIII) from E. coli as a model system the rearrangement of side-chain interactions upon inhibitor binding are identified by comparing amino acid interaction networks for both the R- and T-states of AKIII. Steered molecular dynamics simulation is then applied to study the dynamic conformational change and the energy transduction process, which is followed by identification of co-evolved interaction network involved in the allosteric regulation. To verify the co-evolved allo-network, mutations of AKIII are examined and modulation of the allosteric regulation is demonstrated by site-directed mutagenesis of the key residues. As illustrated, this study proposes a strategy to identify the co-evolved interaction network that drives the allosteric process. The key feature of the strategy is that key residues involved in the energy transfer pathway can be quickly determined by co-evolutionary analysis of the interfacial interactions of motifs.Publicationtype: Journal Article77 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesisL-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered Escherichia coli has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.e. the galactose permease/glucokinase (GalP/Glk) system, can theoretically double the Trp yield from glucose. To explore this possibility, a PTS and GalP/Glk-dependent E. coli strain was constructed from a previously rationally developed Trp producer strain S028. However, the growth rate of the S028 mutant was severely impaired. To overcome this problem, promoter screening for modulated gene expression of GalP/Glk was carried out, following by a batch mode of adaptive laboratory evolution (ALE) which resulted in a strain K3 with a similar Trp yield and concentration as S028. In order to obtain a more efficient Trp producer, a novel continuous ALE system was developed by combining CRISPR/Cas9-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of Trp-mediated fluorescence intensity. With the aid of this automatic system (auto-CGSS), a promising strain T5 was obtained and fed-batch fermentations showed an increase of Trp yield by 19.71% with this strain compared with that obtained by the strain K3 (0.164 vs. 0.137 g/g). At the same time, the specific production rate was increased by 52.93% (25.28 vs. 16.53 mg/g /h). Two previously engineered enzyme variants AroG and AnTrpC were integrated into the strain T5, resulting in a highly productive strain T5AA with a Trp yield of 0.195 g/g and a specific production rate of 28.83 mg/g /h. − D6G−D7A R378F DCW DCWPublicationtype: Journal ArticleScopus© Citations 16 168 526 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Dynamic energy correlation analysis of E. coli aspartokinase III and alteration of allosteric regulation by manipulating energy transduction pathwaysConformational change associated with allosteric regulation in a protein is ultimately driven by energy transformation. However, little is known about the latter process. In this work, we combined steered molecular dynamics simulations and sequence conservation analysis to investigate the conformational changes and energy transformation in the allosteric enzyme aspartokinase III (AK III) from Escherichia coli. Correlation analysis of energy change at residue level indicated significant transformation between electrostatic energy and dihedral angle energy during the allosteric regulation. Key amino acid residues located in the corresponding energy transduction pathways were identified by dynamic energy correlation analysis. To verify their functions, residues with a high energy correlation in the pathways were altered and their effects on allosteric regulation of AKIII were determined. This study sheds new insights into energy transformation during allosteric regulation of AK III and proposes a strategy to identify key residues that are involved in intramolecular energy transduction and thus in driving the allosteric process.Publicationtype: Journal ArticleScopus© Citations 1 132 150 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Engineering of Phosphoserine Aminotransferase Increases the Conversion of l-Homoserine to 4-Hydroxy-2-ketobutyrate in a Glycerol-Independent Pathway of 1,3-Propanediol Production from Glucose(2019-09-01); ; ; ; Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4-hydroxy-2-ketobutyrate, a key reaction in producing 1,3-propanediol (1,3-PDO) from glucose in a novel glycerol-independent metabolic pathway. To this end, a computation-based rational approach is used to change the substrate specificity of SerC from l-phosphoserine to l-homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerCR42W/R77W, is successfully improved by 4.2-fold in comparison to the wild type when l-homoserine is used as the substrate, while its activity toward the natural substrate l-phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3-PDO production, the “homoserine to 1,3-PDO” pathway is constructed in E. coli by coexpression of SerCR42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L−1 1,3-PDO in fed-batch fermentation, which is 13-fold higher than the wild-type strain and represents an important step forward to realize the promise of the glycerol-independent synthetic pathway for 1,3-PDO production from glucose.Publicationtype: Journal Article259 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E. coli for efficient tryptophan bioproduction(2018-05); ; ; The L-tryptophan (Trp) biosynthesis pathway is highly regulated at multiple levels. The three types of regulations identified so far, namely repression, attenuation, and feedback inhibition have greatly impacted our understanding and engineering of cellular metabolism. In this study, feed-forward regulation is discovered as a novel regulation of this pathway and explored for engineering Escherichia coli for more efficient Trp biosynthesis. Specifically, indole glycerol phosphate synthase (IGPS) of the multifunctional enzyme TrpC from E. coli is found to be feed-forward inhibited by anthranilate noncompetitively. Surprisingly, IGPS of TrpC from both Saccharomyces cerevisiae and Aspergillus niger was found to be feed-forward activated, for which the glutamine aminotransferase domain is essential. The anthranilate binding site of IGPS from E. coli is identified and mutated, resulting in more tolerant variants for improved Trp biosynthesis. Furthermore, expressing the anthranilate-activated TrpC from A. niger in a previously engineered Trp producing E. coli strain S028 made the strain more robust in growth and more efficient in Trp production in bioreactor. It not only increased the Trp concentration from 19 to 29 g/L within 42 h, but also improved the maximum Trp yield from 0.15 to 0.18 g/g in simple fed-batch fermentations, setting a new level to rationally designed Trp producing strains. The findings are of fundamental interest for understanding and re-designing dynamics and control of metabolic pathways in general and provide a novel target and solution to engineering of E. coli for efficient Trp production particularly.Publicationtype: Journal Article162 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Engineering Biomolecular Switches for Dynamic Metabolic ControlLiving organisms have been exploited as production hosts for a large variety of compounds. To improve the efficiency of bioproduction, metabolic pathways in an organism are usually manipulated by various genetic modifications. However, bottlenecks during the conversion of substrate to a desired product may result from cellular regulations at different levels. Dynamic regulation of metabolic pathways according to the need of cultivation process is therefore essential for developing effective bioprocesses, but represents a major challenge in metabolic engineering and synthetic biology. To this end, switchable biomolecules which can sense the intracellular concentrations of metabolites with different response types and dynamic ranges are of great interest. This chapter summarizes recent progress in the development of biomolecular switches and their applications for improvement of bioproduction via dynamic control of metabolic fluxes. Further studies of bioswitches and their applications in industrial strain development are also discussed.Publicationtype: Book Part235 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Reengineering substrate specificity of E. coli glutamate dehydrogenase using a position-based prediction methodObjective: To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM). Results: The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg−1. Conclusion: The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.Publicationtype: Journal Article98 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Exploring signal transduction in heteromultimeric protein based on energy dissipation modelDynamic intersubunit interactions are key elements in the regulation of many biological systems. A better understanding of how subunits interact with each other and how their interactions are related to dynamic protein structure is a fundamental task in biology. In this paper, a heteromultimeric allosteric protein, Corynebacterium glutamicum aspartokinase, is used as a model system to explore the signal transduction involved in intersubunit interactions and allosteric communication with an emphasis on the intersubunit signaling process. For this purpose, energy dissipation simulation and network construction are conducted for each subunit and the whole protein. Comparison with experimental results shows that the new approach is able to predict all the mutation sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. Additionally, analysis revealed that the function of the effector threonine is to facilitate the binding of the two subunits without contributing to the allosteric communication. During the allosteric regulation upon the binding of the effector lysine, signals can be transferred from the β-subunit to the catalytic site of the α-subunit through both a direct way of intersubunit signal transduction, and an indirect way: first, to the regulatory region of the α-subunit by intersubunit signal transduction and then to the catalytic region by intramolecular signal transduction. Therefore, the new approach is able to illustrate the diversity of the underlying mechanisms when the strength of feedback inhibition by the effector(s) is modulated, providing useful information that has potential applications in engineering heteromultimeric allosteric regulation.Publicationtype: Journal Article54 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII proteinPII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048-1059. © 2013 The Authors.Publicationtype: Journal ArticleScopus© Citations 8 33