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combinatorial biosynthesis

Ulschan Bathe, Alain Tissier
In plant terpene biosynthesis, oxidation of the hydrocarbon backbone produced by terpene synthases is typically carried out by cytochrome P450 oxygenases (CYPs). The modifications introduced by CYPs include hydroxylations, sequential oxidations at one position and ring rearrangements and closures. These reactions significantly expand the structural diversity of terpenoids, but also provide anchoring points for further decorations by various transferases. In recent years, there has been a significant increase in reports of CYPs involved in plant terpene pathways...
February 5, 2019: Phytochemistry
Shuai Qian, Ye Li, Patrick C Cirino
BACKGROUND: Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict...
January 29, 2019: Microbial Cell Factories
Tian Tian, Jing Wei Kang, Aram Kang, Taek Soon Lee
CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway...
February 5, 2019: ACS Synthetic Biology
Haibo Yuan, Yanfeng Liu, Xueqin Lv, Jianghua Li, Guocheng Du, Zhongping Shi, Long Liu
The compound 2,5-furandicarboxylic acid (FDCA), an important bio-based monomer for the production of various polymers, can be obtained from 5-hydroxymethylfurfural (HMF). However, efficient production of FDCA from HMF via biocatalysis has not been well studied. In this study, we report the identification of key genes that are involved in FDCA synthesis and then the engineering of Raoultella ornithinolytica BF60 for biocatalytic oxidation of HMF to FDCA using its resting cells. Specifically, previously unknown candidate genes, adhP3 and alkR , which were responsible for the reduction of HMF to the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol), were identified by transcriptomic analysis...
December 28, 2018: Journal of Microbiology and Biotechnology
Biplav Shrestha, Ramesh Prasad Pandey, Sumangala Darsandhari, Prakash Parajuli, Jae Kyung Sohng
BACKGROUND: Multi-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector...
January 17, 2019: Microbial Cell Factories
Qian Deng, Liqiongzi Xiao, Yang Liu, Lixin Zhang, Zixin Deng, Changming Zhao
Ivermectin, a kind of valuable derivatives of Avermectin, is distinct from Avermectin due to the saturated bond at C22-C23 position. Combinatorial biosynthesis of Ivermectins based on Avermectins biosynthetic gene cluster ( ave ) has been achieved recently, while the establishment of an Ivermectin homogeneous component producing strain is challenging because of the limited compatibility between the native and heterologous polyketide synthase (PKS) domains. In this study, the PKS module 2 Dehydratase (DH)-Enoylreductase (ER)-Ketoreductase (KR) domain set of Meilingmycin, which is another naturally occurring homologue of Avermectin, was employed to substitute the DH-KR domains of Avermectins PKS module 2 to generate an Ivermectin biosynthetic gene cluster ( ive )...
March 2019: Synthetic and Systems Biotechnology
Kei Sato, Yohei Katsuyama, Kousuke Yokota, Takayoshi Awakawa, Takeaki Tezuka, Yasuo Ohnishi
Fogacin and two novel fogacin derivatives, fogacins B and C, were isolated from the rare actinomycete Actinoplanes missouriensis. Biosynthesis of fogacin C apparently requires β-alkylation of a polyketide chain. In fact, the fogacin biosynthetic type II polyketide synthase (PKS) gene cluster contains a hydroxymethylglutaryl-CoA synthase (HCS) cassette, which is usually responsible for β-alkylation in the type I PKS system. Another characteristic of the fog cluster is that it encodes two sets of ketosynthase (KS) and chain length factor (CLF)...
December 16, 2018: Chembiochem: a European Journal of Chemical Biology
Ying Zhao, Bi-Han Wu, Zhen-Ning Liu, Jianjun Qiao, Guang-Rong Zhao
Resveratrol, a plant-derived polyphenolic compound with various health activities, is widely used in nutraceutical and food additives. Herein, combinatorial optimization of resveratrol biosynthetic pathway and intracellular environment of E. coli was carried out. Screening pathway genes from various species and exploring their expression pattern, we constructed initial resveratrol-producing strain. Further targeting at availability of malonyl-CoA through expressing ACC of Corynebacterium glutamicum and antisense inhibiting native fabD significantly increased resveratrol biosynthesis...
November 29, 2018: Journal of Agricultural and Food Chemistry
Yujie Yuan, Martin Litzenburger, Shu Cheng, Guangkai Bian, Ben Hu, Pan Yan, Yousheng Cai, Zixin Deng, Rita Bernhardt, Tiangang Liu
Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core-skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors for the structure diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions...
November 28, 2018: Chembiochem: a European Journal of Chemical Biology
Nicholas Gold, Elena Fossati, Cecilie Cetti Hansen, Marcos Di Falco, Veronique Douchin, Vincent J J Martin
Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologs were tested in batch growth...
November 26, 2018: ACS Synthetic Biology
Rishab Ramapriyan, Mauricio S Caetano, Hampartsoum B Barsoumian, Ana Carolina P Mafra, Erika Pereira Zambalde, Hari Menon, Efrosini Tsouko, James W Welsh, Maria Angelica Cortez
Many metabolic alterations, including the Warburg effect, occur in cancer cells that influence the tumor microenvironment, including switching to glycolysis from oxidative phosphorylation, using opportunistic modes of nutrient acquisition, and increasing lipid biosynthesis. The altered metabolic landscape of the tumor microenvironment can suppress the infiltration of immune cells and other functions of antitumor immunity through the production of immune-suppressive metabolites. Metabolic dysregulation in cancer cells further affects the expression of cell surface markers, which interferes with immune surveillance...
November 12, 2018: Pharmacology & Therapeutics
Hiroya Itoh, Makoto Matsui, Yuki Miyamura, Itaru Takeda, Jun Ishii, Toshitaka Kumagai, Masayuki Machida, Takashi Shibata, Masanori Arita
For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes...
November 13, 2018: ACS Synthetic Biology
Yuki Tobimatsu, Mathias Schuetz
The final step of lignin biosynthesis is the polymerization of monolignols in apoplastic cell wall domains. In this process, monolignols secreted by lignifying cells, or occasionally neighboring non-lignifying and/or other lignifying cells, are activated by cell-wall-localized oxidation systems, such as laccase/O2 and/or peroxidase/H2 O2 , for combinatorial radical coupling to make the final lignin polymers. Plants can precisely control when, where, and which types of lignin polymers are assembled at tissue and cellular levels, but do not control the polymers' exact chemical structures per se...
October 22, 2018: Current Opinion in Biotechnology
Philip A Storm, Paramita Pal, Callie R Huitt-Roehl, Craig A Townsend
Polyketide C-methylation occurs during a programmed sequence of dozens of reactions carried out by multidomain polyketide synthases (PKSs). Fungal PKSs perform these reactions iteratively, where a domain may be exposed to and act upon multiple enzyme-tethered intermediates during biosynthesis. We surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs to gain insight into how different methylation patterns are installed. Our in vitro results show that control of methylation resides primarily with the CMeT, and CMeTs can intercept and methylate intermediates from noncognate nonreducing PKS domains...
October 30, 2018: ACS Chemical Biology
Alexander G Ekström, Jue Theresa Wang, Juraj Bella, Dominic J Campopiano
Dynamic combinatorial chemistry (DCC) is a powerful tool to identify ligands for biological targets. We used 19F NMR as an in situ, non-invasive technique for measuring the composition of a dynamic combinatorial library (DCL) of N-acylhydrazones (NAHs). An NAH DCL, constructed from a fluoro-aromatic aldehyde and a small set of hydrazides, was targetted at ecFabH, an essential enzyme in bacterial fatty acid biosynthesis. Our NMR analysis identified a tert-butyl NAH as the best binder which was confirmed by enzymatic assay...
October 18, 2018: Organic & Biomolecular Chemistry
Fu Yan, Christian Burgard, Alexander Popoff, Nestor Zaburannyi, Gregor Zipf, Josef Maier, Hubert S Bernauer, Silke C Wenzel, Rolf Müller
Synthetic biology techniques coupled with heterologous secondary metabolite production offer opportunities for the discovery and optimisation of natural products. Here we developed a new assembly strategy based on type IIS endonucleases and elaborate synthetic DNA platforms, which could be used to seamlessly assemble and engineer biosynthetic gene clusters (BGCs). By applying this versatile tool, we designed and assembled more than thirty different artificial myxochromide BGCs, each around 30 kb in size, and established heterologous expression platforms using a derivative of Myxococcus xanthus DK1622 as a host...
October 14, 2018: Chemical Science
Elizabeth Skellam
Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts...
October 10, 2018: Trends in Biotechnology
Haibo Yuan, Yanfeng Liu, Xueqin Lv, Jianghua Li, Guocheng Du, Zhongping Shi, Long Liu
The compound 2,5-furandicarboxylic acid (FDCA), an important bio-based monomer for the production of various polymers, can be obtained from 5-hydroxymethylfurfural (HMF). However, efficient production of FDCA from HMF via biocatalysis has not been well studied. In this study, we report the identification of key genes that are involved in FDCA synthesis and then the engineering of Raoultella ornithinolytica BF60 for biocatalytic oxidation of HMF to FDCA using its resting cells. Specifically, previously unknown candidate genes, adhP3 and alkR , which were responsible for the reduction of HMF to the undesired product 2,5-bis(hydroxymethyl) furan (HMF alcohol), were identified by transcriptomic analysis...
October 10, 2018: Journal of Microbiology and Biotechnology
A C Ward, Nee Allenby
The need for new antimicrobials is indisputable. The flight from natural products in drug discovery was unfortunate. However, the revolution that is genome mining, enabled by the explosion in sequencing technology, is a cause for hope. Nevertheless, renewed search and discovery is still a challenge. We explore novel metabolite diversity and the challenges in Streptomyces. Estimating novel bioactive metabolites remaining to be discovered is an important driver for future investment. Frequent re-discovery of known natural products was a major factor in big pharma exiting search and discovery and remains a reality...
September 27, 2018: FEMS Microbiology Letters
Young Jin Ko, Young-Chul Joo, Jeong Eun Hyeon, Eunhye Lee, Myeong-Eun Lee, Jiho Seok, Seung Wook Kim, Chulhwan Park, Sung Ok Han
Zn-porphyrin is a promising organic photosensitizer in various fields including solar cells, interface and biomedical research, but the biosynthesis study has been limited, probably due to the difficulty of understanding complex biosynthesis pathways. In this study, we developed a Corynebacterium glutamicum platform strain for the biosynthesis of Zn-coproporphyrin III (Zn-CP III), in which the heme biosynthesis pathway was efficiently upregulated. The pathway was activated and reinforced by strong promoter-induced expression of hemAM (encoding mutated glutamyl-tRNA reductase) and hemL (encoding glutamate-1-semialdehyde aminotransferase) genes...
September 27, 2018: Scientific Reports
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