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Li L.,CAS Shanghai Institutes for Biological Sciences | Li L.,University of Chinese Academy of Sciences | Jiang W.,CAS Shanghai Institutes for Biological Sciences | Jiang W.,Jiangsu National Synergetic Innovation Center for Advanced Materials | And 2 more authors.
Biotechnology Advances | Year: 2017

With the rapidly growing number of sequenced microbial (meta)genomes, enormous cryptic natural product (NP) biosynthetic gene clusters (BGCs) have been identified, which are regarded as a rich reservoir for novel drug discovery. A series of powerful tools for engineering BGCs has accelerated the discovery and development of pharmaceutically active NPs. Here, we describe recent advances in the strategies for BGCs manipulation, which are driven by emerging technologies, including efficient DNA recombination systems, versatile CRISPR/Cas9 genome editing tools and diverse DNA assembly methods. We further discuss how these approaches could be used for genome mining studies and industrial strain improvement. © 2017 Elsevier Inc.


Li Q.,East China University of Science and Technology | Huang B.,East China University of Science and Technology | Wu H.,East China University of Science and Technology | Li Z.,East China University of Science and Technology | And 2 more authors.
Bioresource Technology | Year: 2017

Glycerol is an important resource for production of value-added bioproducts due to its large availability from the biodiesel industry as a by-product. In this study, two metabolic regulation strategies were applied in the aerobic stage of a two-stage fermentation to achieve high metabolic capacities of the pflB ldhA double mutant Escherichia coli strain overexpressing phosphoenolpyruvate carboxykinase (PCK) in the subsequent anaerobic stage: use of acetate as a co-carbon source of glycerol and restriction of oxygen supply in the PCK induction period. The succinate concentration achieved 926.7 mM with a yield of 0.91 mol/mol during the anaerobic stage of fermentation in a 1.5-L reactor. qRT-PCR indicated that the two strategies enhanced transcription of genes related with glycerol metabolism and succinate production. Our results showed this metabolically engineered E. coli strain has a great potential in producing succinate using glycerol as carbon source. © 2017 Elsevier Ltd


Jiang Y.,CAS Shanghai Institutes for Biological Sciences | Chen B.,CAS Shanghai Institutes for Biological Sciences | Duan C.,CAS Shanghai Institutes for Biological Sciences | Sun B.,CAS Shanghai Institutes for Biological Sciences | And 3 more authors.
Applied and Environmental Microbiology | Year: 2015

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%. © 2015, American Society for Microbiology.


Li L.,CAS Shanghai Institutes for Biological Sciences | Zhao Y.,CAS Shanghai Institutes for Biological Sciences | Ruan L.,Shanghai Laiyi Center for Biopharmaceuticals R and D | Yang S.,CAS Shanghai Institutes for Biological Sciences | And 4 more authors.
Metabolic Engineering | Year: 2015

Pristinamycin, which is a streptogramin antibiotic produced by Streptomyces pristinaespiralis, contains two chemically unrelated compounds, pristinamycin I (PI) and pristinamycin II (PII). Semi-synthetic derivatives of PI and PII have been approved for use in human medicine to treat a broad range of drug-resistant pathogens. In this study, we design and implement a combinatorial metabolic engineering strategy for improving PII production. First, an extra copy of the PII biosynthetic gene cluster, which was assembled using a modified Gibson assembly method for cloning large DNA fragments with high GC contents, was introduced into a high-producing strain S. pristinaespiralis HCCB10218. This duplication of the PII biosynthetic gene cluster resulted in a maximum increase in PII titer by 45%. Second, all seven cluster-situated regulatory genes (from papR1 to papR6 and spbR) were systematically manipulated. Higher PII titers were achieved by deleting either one of the two repressor genes papR3 or papR5 in combination with overexpression of both activator genes papR4 and papR6, and the resulting strains {increment}. papR3+. R4R6 and {increment}. papR5+. R4R6 showed maximum increases in PII production by 99% and 75%, respectively. A combination of the above two different approaches was employed. Integration of the assembled PII gene cluster (BAC-F1F15) into {increment}. papR5+. R4R6 led to the highest PII titer improvement, which was approximately 1.5-fold higher than the parental strain. By adding the macroreticular resin, which can separate pristinamycin in situ and thereby lessen end-product feedback inhibition and toxic effects, PII titers of the final engineered strain {increment}. papR5+. R4R6/BAC-F1F15 reached 1.13 and 1.16. g/L in the Erlenmeyer flask and 5-L bioreactor, respectively, with 5.13- and 5.26-fold improvements over the parental strain. Taken together, this combinatorial strategy is an efficient method to optimize PII biosynthesis of S. pristinaespiralis and may be extended to other industrially used streptomycetes for strain improvement. © 2015 International Metabolic Engineering Society.


Dun J.,CAS Shanghai Institutes for Biological Sciences | Zhao Y.,CAS Shanghai Institutes for Biological Sciences | Zheng G.,CAS Shanghai Institutes for Biological Sciences | Zhu H.,CAS Shanghai Institutes for Biological Sciences | And 6 more authors.
Journal of Bacteriology | Year: 2015

There are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster of Streptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of the papR6 gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, either papR6 deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region of snaF, the first gene of the snaFE1E2GHIJK operon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C11 αβ-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5'-GAGG-4 nt-CCTC-3') was identified. Transcriptional analysis showed that inactivation of the papR6 gene led to markedly decreased expression of snaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ΔpapR6 strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C11 αβ-unsaturated thioester. © 2015, American Society for Microbiology.


Sun Z.,CAS Shanghai Institutes for Biological Sciences | Chen Y.,CAS Shanghai Institutes for Biological Sciences | Yang C.,CAS Shanghai Institutes for Biological Sciences | Yang S.,CAS Shanghai Institutes for Biological Sciences | And 4 more authors.
Molecular Microbiology | Year: 2015

Summary: d-Xylose is the most abundant fermentable pentose in nature and can serve as a carbon source for many bacterial species. Since d-xylose constitutes the major component of hemicellulose, its metabolism is important for lignocellulosic biomass utilization. Here, we report a six-protein module for d-xylose signaling, uptake and regulation in solvent-producing Clostridium beijerinckii. This module consists of a novel 'three-component system' (a putative periplasmic ABC transporter substrate-binding protein XylFII and a two-component system LytS/YesN) and an ABC-type d-xylose transporter XylFGH. Interestingly, we demonstrate that, although XylFII harbors a transmembrane domain, it is not involved in d-xylose transport. Instead, XylFII acts as a signal sensor to assist the response of LytS/YesN to extracellular d-xylose, thus enabling LytS/YesN to directly activate the transcription of the adjacent xylFGH genes and thereby promote the uptake of d-xylose. To our knowledge, XylFII is a novel single transmembrane sensor that assists two-component system to respond to extracellular sugar molecules. Also of significance, this 'three-component system' is widely distributed in Firmicutes, indicating that it may play a broad role in this bacterial phylum. The results reported here provide new insights into the regulatory mechanism of d-xylose sensing and transport in bacteria. © 2014 John Wiley & Sons Ltd.


Hu G.,East China University of Science and Technology | Hu G.,Shanghai Collaborative Innovation Center for Biomanufacturing Technology | Yu W.,East China University of Science and Technology
Food Chemistry | Year: 2015

The paper study the functional properties of hemicellulose B (RBHB) and rice bran insoluble dietary fibre (RBDF) to develop an acceptable low fat meat product enriched with high content fibre from defatted rice bran. Meatballs were produced with three different formulations including 2%, 4% and 6% RBHB or RBDF addition. The total trans fatty acids were lower and the ratio of total unsaturated fatty acids to total saturated fatty acids was higher in the samples with added RBHB than in the control meatballs. Meatballs containing RBHB had lower concentrations of total fat and total trans fatty acids than the control samples. Sensory evaluations revealed that meatballs with 2%, 4% and 6% RBHB were overall acceptable. This confirms that the RBHB preparation from defatted rice bran has great potential in food applications, especially in development of functional foods including functional meat products. © 2014 Elsevier Ltd.All rights reserved.


Sun F.,East China University of Science and Technology | Zhu F.,East China University of Science and Technology | Shao X.,East China University of Science and Technology | Li Z.,East China University of Science and Technology | Li Z.,Shanghai Collaborative Innovation Center for Biomanufacturing Technology
Synlett | Year: 2015

An efficient and versatile method was developed for the access of multisubstituted 1,8-naphthyridine derivatives through a one-pot, three-component protocol from heterocyclic ketene animals, malononitrile dimer, and aryl aldehydes in high yields. The 1,8-naphthyridines were formed via Knoevenagel condensation, aza-ene reaction, imine-enamine tautomerization, and intramolecular cyclization. © Georg Thieme Verlag Stuttgart New York.


Yi Y.,East China University of Science and Technology | Sheng H.,East China University of Science and Technology | Li Z.,East China University of Science and Technology | Li Z.,Shanghai Collaborative Innovation Center for Biomanufacturing Technology | Ye Q.,East China University of Science and Technology
BMC Biotechnology | Year: 2014

Background: Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production.Result: In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate.Conclusions: The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt. © 2014 Yi et al.; licensee BioMed Central Ltd.


Zhang C.,East China University of Science and Technology | Zhang C.,Chalmers University of Technology | Ji B.,Chalmers University of Technology | Mardinoglu A.,Chalmers University of Technology | And 3 more authors.
Bioinformatics | Year: 2015

Motivation: In recent years, genome-scale metabolic models (GEMs) have played important roles in areas like systems biology and bioinformatics. However, because of the complexity of gene- reaction associations, GEMs often have limitations in gene level analysis and related applications. Hence, the existing methods were mainly focused on applications and analysis of reactions and metabolites. Results: Here, we propose a framework named logic transformation of model (LTM) that is able to simplify the gene-reaction associations and enables integration with other developed methods for gene level applications. We show that the transformed GEMs have increased reaction and metabolite number as well as degree of freedom in flux balance analysis, but the gene-reaction associations and the main features of flux distributions remain constant. In addition, we develop two methods, OptGeneKnock and FastGeneSL by combining LTM with previously developed reaction-based methods. We show that the FastGeneSL outperforms exhaustive search. Finally, we demonstrate the use of the developed methods in two different case studies. We could design fast genetic intervention strategies for targeted overproduction of biochemicals and identify double and triple synthetic lethal gene sets for inhibition of hepatocellular carcinoma tumor growth through the use of OptGeneKnock and FastGeneSL, respectively. © The Author 2015. Published by Oxford University Press. All rights reserved.

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