Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

Abstract Background The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. Conclusions In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.

Download Full-text

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Microbial Cell Factories ◽

10.1186/s12934-021-01615-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Zhenning Liu ◽

Xue Zhang ◽

Dengwei Lei ◽

Bin Qiao ◽

Guang-Rong Zhao

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Phosphopantetheinyl Transferase ◽

Chromosome Engineering ◽

Microbial Cell Factory ◽

E Coli ◽

Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Download Full-text

Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids in Escherichia coli

10.1101/2020.03.03.974378 ◽

2020 ◽

Author(s):

Lixia Fang ◽

Jie Fan ◽

Congya Wang ◽

Yingxiu Cao ◽

Hao Song

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Free Fatty Acids ◽

Metabolic Networks ◽

High Titer ◽

Cell Structure ◽

Cell Factory ◽

Microbial Cell Factory ◽

Cell Potential ◽

E Coli

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cell potential via identifying and engineering beneficial gene targets in the sophisticated metabolic networks. Here, we develop an approach that integrates CRISPR interference (CRISPRi) to readily modulate genes expression and omics analyses to identify potential targets in multiple cellular processes, enabling systematical discovery of beneficial chromosomal gene targets that can be engineered to optimize free fatty acids (FFAs) production in Escherichia coli. We identify 56 beneficial genes via synergistic CRISPRi-Omics strategy, including 46 novel targets functioning in cell structure and division, and signaling transduction that efficiently facilitate FFAs production. Upon repressing ihfA and overexpressing aidB and tesA’ in E. coli, the recombinant strain LihfA-OaidB results in a FFAs titer of 21.6 g L-1 in fed-batch fermentation, which, to our best knowledge, is the maximum FFAs titer by the recombinant E. coli reported to date.

Download Full-text

Metabolic engineering Escherichia coli for efficient production of icariside D2

Biotechnology for Biofuels ◽

10.1186/s13068-019-1601-x ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Xue Liu ◽

Lingling Li ◽

Jincong Liu ◽

Jianjun Qiao ◽

Guang-Rong Zhao

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Carbon Sources ◽

Gene Expression Pattern ◽

Fine Tuning ◽

Cell Factory ◽

Efficient Production ◽

Uridine Diphosphate ◽

Microbial Cell Factory ◽

E Coli

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Download Full-text

Engineering Rhodosporidium toruloides for limonene production

Biotechnology for Biofuels ◽

10.1186/s13068-021-02094-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Sasa Liu ◽

Mengyao Zhang ◽

Yuyao Ren ◽

Guojie Jin ◽

Yongsheng Tao ◽

...

Keyword(s):

Rhodosporidium Toruloides ◽

Cell Factory ◽

Mevalonate Kinase ◽

Chimeric Enzyme ◽

Methanosarcina Mazei ◽

Limonene Synthase ◽

A Cell ◽

Coa Reductase ◽

Geranyl Pyrophosphate Synthase ◽

Geranyl Pyrophosphate

Abstract Background Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. Results By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. Conclusion In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.

Download Full-text

Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

Bioresources and Bioprocessing ◽

10.1186/s40643-020-00342-6 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Wan-Wen Ting ◽

Shih-I Tan ◽

I-Son Ng

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Rna Polymerase ◽

Green Fluorescence Protein ◽

T7 Rna Polymerase ◽

Cell Factory ◽

T7 Promoter ◽

Iptg Induction ◽

E Coli ◽

A Cell

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

Download Full-text

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽

10.1093/genetics/166.2.661 ◽

2004 ◽

Vol 166 (2) ◽

pp. 661-668

Author(s):

Mandy Kim ◽

Erika Wolff ◽

Tiffany Huang ◽

Lilit Garibyan ◽

Ashlee M Earl ◽

...

Keyword(s):

Dna Sequences ◽

Deinococcus Radiodurans ◽

Genetic System ◽

Base Change ◽

Base Substitution ◽

Wild Type ◽

Base Pairs ◽

E Coli ◽

Radiation Induced ◽

Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

Download Full-text

Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids

Nature Communications ◽

10.1038/s41467-021-25243-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lixia Fang ◽

Jie Fan ◽

Shulei Luo ◽

Yaru Chen ◽

Congya Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Free Fatty Acids ◽

Stress Responses ◽

Metabolic Networks ◽

Target Identification ◽

High Titer ◽

Cell Factory ◽

Microbial Cell Factory ◽

Ffa Metabolism

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

Download Full-text

Crystal Structure ofDeinococcus radioduransRecQ Helicase Catalytic Core Domain: The Interdomain Flexibility

BioMed Research International ◽

10.1155/2014/342725 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Sheng-Chia Chen ◽

Chi-Hung Huang ◽

Chia Shin Yang ◽

Tzong-Der Way ◽

Ming-Chung Chang ◽

...

Keyword(s):

Crystal Structure ◽

Deinococcus Radiodurans ◽

Domain Architecture ◽

Genome Integrity ◽

Catalytic Core Domain ◽

Catalytic Core ◽

Core Domain ◽

Winged Helix ◽

Dna Helicases ◽

E Coli

RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ fromDeinococcus radiodurans(DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of theE. coliRecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

Download Full-text

Reassessing Escherichia coli as a cell factory for biofuel production

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2017.02.010 ◽

2017 ◽

Vol 45 ◽

pp. 92-103 ◽

Cited By ~ 28

Author(s):

Chonglong Wang ◽

Brian F Pfleger ◽

Seon-Won Kim

Keyword(s):

Escherichia Coli ◽

Biofuel Production ◽

Cell Factory ◽

A Cell

Download Full-text

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699235 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuxiao Xie ◽

Shulin Chen ◽

Xiaochao Xiong

Keyword(s):

Yarrowia Lipolytica ◽

Fold Increase ◽

Genetically Engineered ◽

Cell Factory ◽

Yeast Nitrogen Base ◽

Nitrogen Base ◽

Yeast Extract Peptone Dextrose ◽

Dry Cell Weight ◽

A Cell ◽

Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Download Full-text