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1.
Synth Syst Biotechnol ; 5(4): 333-342, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33102829

RESUMO

Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production. However, the direct application of this strain suffers from the transformation inefficiency and plasmid instability. Herein, we conducted genetic modifications at a serial of loci of MG1655 genome, generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways. Using JW128 as the host, the titer of isobutanol reached 5.76 g/L in shake-flask fermentation, and the titer of lycopene reached 1.91 g/L in test-tube fermentation, 40-fold and 5-fold higher than that of original MG1655, respectively. These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.

2.
Int J Syst Evol Microbiol ; 70(10): 5460-5466, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32894215

RESUMO

A bacterial strain, BIT-d1T, was isolated from the gut of plastic-eating larvae of the coleopteran insect Zophobas atratus. Its taxonomic position was analysed using a polyphasic approach. Cells were white-pigmented, Gram-stain-negative, non-motile, long rods without flagella. The 16S rRNA gene sequence (1401 bp) of strain BIT-d1T showed highest similarity (98.0%) to Myroides pelagicus SM1T and 96.6~92.6 % similarity to the other species of the genus Myroides. The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of six housekeeping genes (gyrB, dnaK, tuf, murG, atpA and glyA) and genome sequences, placed strain BIT-d1T in a separate lineage among the genus Myroides, family Flavobacteriaceae. The major isoprenoid quinone was menaquinone-6 (MK-6) and the major fatty acids were C15 : 0 iso, C17 : 0 iso 3-OH and summed feature 9 (comprising iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), which were similar to other members in the genus Myroides. In silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests exhibited the genotypic and phenotypic differentiation of strain BIT-d1T from the other members of the genus Myroides. Therefore, strain BIT-d1T is considered to represent a novel species within the genus Myroides, for which the name Myroides albus sp. nov is proposed. The type strain is BIT-d1T (=CGMCC 1.17043T=KCTC 72447T).


Assuntos
Besouros/microbiologia , Flavobacteriaceae/classificação , Trato Gastrointestinal/microbiologia , Filogenia , Plásticos , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Flavobacteriaceae/isolamento & purificação , Microbioma Gastrointestinal , Genes Bacterianos , Larva , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
3.
Int J Syst Evol Microbiol ; 70(9): 4951-4959, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32744988

RESUMO

A bacterial strain, BIT-B35T, was isolated from the gut of plastic-eating larvae of the Coleoptera insect Zophobas atratus. Its taxonomic position was determined by using a polyphasic approach. Cells were white-pigmented, Gram-stain-negative, motile short rods with terminal flagella. The 16S rRNA gene sequence (1411 bp) of strain BIT-B35T showed highest similarity (98.1%) to Escherichia fergusonii ATCC 35469T and Citrobacter koseri LMG 5519T. The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of seven housekeeping genes (atpD, gyrB, infB, rpoB, pyrG, fusA and leuS) and genome sequences, placed strain BIT-B35T in a separate lineage among the family of Enterobacteriaceae. The major fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The genomic DNA G+C content of strain BIT-B35T was 57.1 mol%. The chemotaxonomic data plus results of physiological and biochemical tests also distinguished strain BIT-B35T from members of other genera within the family Enterobacteriaceae. Therefore, strain BIT-B35T is considered to represent a novel species of a novel genus within the family Enterobacteriaceae, for which the name Intestinirhabdus alba gen. nov., sp. nov. is proposed. The type strain is BIT-B35T (=CGMCC 1.17042T=KCTC 72448T).


Assuntos
Besouros/microbiologia , Enterobacteriaceae/classificação , Microbioma Gastrointestinal , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Enterobacteriaceae/isolamento & purificação , Ácidos Graxos/química , Genes Bacterianos , Larva/microbiologia , Plásticos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
4.
ACS Synth Biol ; 9(9): 2214-2227, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786348

RESUMO

Triterpenoids represent a diverse group of phytochemicals that are widely distributed in the plant kingdom and have many biological activities. The heterologous production of triterpenoids in Saccharomyces cerevisiae has been successfully implemented by introducing various triterpenoid biosynthetic pathways. By engineering related enzymes as well as through yeast metabolism, the yield of various triterpenoids is significantly improved from the milligram per liter scale to the gram per liter scale. This achievement demonstrates that engineering critical enzymes is considered a potential strategy to overcome the main hurdles of the industrial application of these potent natural products. Here, we review strategies for designing enzymes to improve the yield of triterpenoids in S. cerevisiae in terms of three main aspects: 1, elevating the supply of the precursor 2,3-oxidosqualene; 2, optimizing triterpenoid-involved reactions; and 3, lowering the competition of the native sterol pathway. Then, we provide challenges and prospects for further enhancing triterpenoid production in S. cerevisiae.

5.
Appl Microbiol Biotechnol ; 104(18): 7943-7956, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32794018

RESUMO

Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering. KEY POINTS: • This article reports an efficient genome engineering tool for E. coli. • The tool is advantageous for the manipulations of long DNA fragments. • The tool has been successfully applied for genome simplification. • The tool has been successfully applied for metabolic engineering.

6.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32718035

RESUMO

Serotyping has traditionally been considered the basis for surveillance of Salmonella, but it cannot distinguish distinct lineages sharing the same serovar that vary in host range, pathogenicity and epidemiology. However, polyphyletic serovars have not been extensively investigated. Public health microbiology is currently being transformed by whole-genome sequencing (WGS) data, which promote the lineage determination using a more powerful and accurate technique than serotyping. The focus in this study is to survey and analyze putative polyphyletic serovars. The multi-locus sequence typing (MLST) phylogenetic analysis identified four putative polyphyletic serovars, namely, Montevideo, Bareilly, Saintpaul, and Muenchen. Whole-genome-based phylogeny and population structure highlighted the polyphyletic nature of Bareilly and Saintpaul and the multi-lineage nature of Montevideo and Muenchen. The population of these serovars was defined by extensive genetic diversity, the open pan genome and the small core genome. Source niche metadata revealed putative existence of lineage-specific niche adaptation (host-preference and environmental-preference), exhibited by lineage-specific genomic contents associated with metabolism and transport. Meanwhile, differences in genetic profiles relating to virulence and antimicrobial resistance within each lineage may contribute to pathogenicity and epidemiology. The results also showed that recombination events occurring at the H1-antigen loci may be an important reason for polyphyly. The results presented here provide the genomic basis of simple, rapid, and accurate identification of phylogenetic lineages of these serovars, which could have important implications for public health.

7.
Microb Cell Fact ; 19(1): 63, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156270

RESUMO

BACKGROUND: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. RESULTS: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. CONCLUSIONS: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia/genética , Recombinação Genética , Deleção de Sequência
8.
Microb Cell Fact ; 19(1): 79, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32220254

RESUMO

BACKGROUND: Owing to the increase in energy consumption, fossil fuel resources are gradually depleting which has led to the growing environmental concerns; therefore, scientists are being urged to produce sustainable and ecofriendly fuels. Thus, there is a growing interest in the generation of biofuels from renewable energy resources using microbial fermentation. MAIN TEXT: Butanol is a promising biofuel that can substitute for gasoline; unfortunately, natural microorganisms pose challenges for the economical production of 1-butanol at an industrial scale. The availability of genetic and molecular tools to engineer existing native pathways or create synthetic pathways have made non-native hosts a good choice for the production of 1-butanol from renewable resources. Non-native hosts have several distinct advantages, including using of cost-efficient feedstock, solvent tolerant and reduction of contamination risk. Therefore, engineering non-native hosts to produce biofuels is a promising approach towards achieving sustainability. This paper reviews the currently employed strategies and synthetic biology approaches used to produce 1-butanol in non-native hosts over the past few years. In addition, current challenges faced in using non-native hosts and the possible solutions that can help improve 1-butanol production are also discussed. CONCLUSION: Non-native organisms have the potential to realize commercial production of 1- butanol from renewable resources. Future research should focus on substrate utilization, cofactor imbalance, and promoter selection to boost 1-butanol production in non-native hosts. Moreover, the application of robust genetic engineering approaches is required for metabolic engineering of microorganisms to make them industrially feasible for 1-butanol production.


Assuntos
1-Butanol/metabolismo , Engenharia Genética/métodos
9.
Biotechnol Biofuels ; 13: 29, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32127916

RESUMO

Background: Protein-based bioconversion has been demonstrated as a sustainable approach to produce higher alcohols and ammonia fertilizers. However, owing to the switchover from transcription mediated by the bacterial RNA polymerase σ70 to that mediated by alternative σ factors, the biofuel production driven by σ70-dependent promoters declines rapidly once cells enter the stationary phase or encounter stresses. To enhance biofuel production, in this study the growth phase-independent and nitrogen-responsive transcriptional machinery mediated by the σ54 is exploited to drive robust protein-to-fuel conversion. Results: We demonstrated that disrupting the Escherichia coli ammonia assimilation pathways driven by glutamate dehydrogenase and glutamine synthetase could sustain the activity of σ54-mediated transcription under ammonia-accumulating conditions. In addition, two σ54-dependent promoters, argTp and glnAp2, were identified as suitable candidates for driving pathway expression. Using these promoters, biofuel production from proteins was shown to persist to the stationary phase, with the net production in the stationary phase being 1.7-fold higher than that derived from the optimal reported σ70-dependent promoter P LlacO1. Biofuel production reaching levels 1.3- to 3.4-fold higher than those of the σ70-dependent promoters was also achieved by argTp and glnAp2 under stressed conditions. Moreover, the σ54-dependent promoters realized more rapid and stable production than that of σ70-dependent promoters during fed-batch fermentation, producing up to 4.78 g L - 1 of total biofuels. Conclusions: These results suggested that the nitrogen-responsive transcriptional machinery offers the potential to decouple production from growth, highlighting this system as a novel candidate to realize growth phase-independent and stress-resistant biofuel production.

10.
Int J Syst Evol Microbiol ; 70(2): 790-796, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31675291

RESUMO

A bacterial strain, BIT-26T, was isolated from the gut of plastic-eating mealworm Tenebrio molitor L. The taxonomic position of this new isolate was investigated by using a polyphasic approach. Cells of the strain were Gram-stain-negative, facultatively anaerobic, motile rods with peritrichous flagella. The 16S rRNA gene sequence (1412 bp) of strain BIT-26T showed the highest similarity (97.4 %) to Erwinia piriflorinigrans CFBP 5888T, followed by Citrobacter sedlakii NBRC 105722T (97.3 %), Mixta calida LMG 25383T (97.3 %), Cronobacter muytjensii ATCC 51329T (97.2 %) and Mixta theicola QC88-366 T (97.2 %). The results of phylogenetic analyses, based on the 16S rRNA gene and concatenated sequences of four housekeeping genes (atpD, gyrB, infB and rpoB), placed strain BIT-26T within the genus Mixta of the family Erwiniaceae. This affiliation was also supported by the chemotaxonomic data. Strain BIT-26T had similar predominant fatty acids, including C12 : 0, C14 : 0, C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c, to species of the genus Mixta. In silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain BIT-26T from other species of the genus Mixta with validly published names. Therefore, strain BIT-26T is considered to represent a novel species, for which the name Mixta tenebrionis sp. nov is proposed. The type strain is BIT-26T (=CGMCC 1.17041T=KCTC 72449T).


Assuntos
Gammaproteobacteria/classificação , Trato Gastrointestinal/microbiologia , Filogenia , Plásticos , Tenebrio/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Gammaproteobacteria/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
11.
Sheng Wu Gong Cheng Xue Bao ; 35(12): 2227-2237, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31880132

RESUMO

Enzymes are widely used in medical and biopharmaceuticals. They can be used not only for various disease treatments, but also clinical diagnosis. The use of microorganisms to express heterologous proteins has become the easiest and fastest way to obtain enzymes. In order to obtain high concentration and high-quality heterologous proteins, a common method is codon optimization of gene sequences. The traditional codon optimization strategy is mainly based on codon bias and GC content, ignoring complex and varied factors such as translational dynamics and metabolic levels. We provide here comprehensive codon optimization strategy based on gene level, transcriptional level, translational level, post-translational level and metabolic level, mainly including codon bias, codon harmonization, codon sensitivity, adjustment of gene sequence structure and some other influencing factors. We also summarize the aspects of strategy content, theoretical support and application. Besides, the advantages and disadvantages of each strategy are also systematically compared, providing an all-round, multi-level and multi-selection optimization strategy for heterogeneous protein expression, and also providing references for the enzyme industry and biopharmaceuticals.


Assuntos
Códon , Composição de Bases
12.
Microb Biotechnol ; 12(6): 1476-1486, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31578818

RESUMO

Synthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5' end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5' end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through ß-galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8-fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine-tuning of gene expression.


Assuntos
Elementos Facilitadores Genéticos , Expressão Gênica , Genética Microbiana/métodos , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas , Fusão Gênica Artificial , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Transcrição Genética
13.
Appl Microbiol Biotechnol ; 103(20): 8497-8509, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31501938

RESUMO

Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. coli) rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in genetic engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Engenharia Genética/métodos , Genética Microbiana/métodos
14.
Metab Eng ; 56: 28-38, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31449878

RESUMO

The wild-type transcription factors are sensitive to their corresponding signal molecules. Using wild-type transcription factors as biosensors to screen industrial overproducers are generally impractical because of their narrow detection ranges. This study took transcription factor BmoR as an example and aimed to expand the detection range of BmoR for screening alcohols overproducers. Firstly, a BmoR mutation library was established, and the mutations distributed randomly in all predicted functional domains of BmoR. Structure of BmoR-isobutanol complex were modelled, and isobutanol binding sites were confirmed by site-directed mutagenesis. Subsequently, the effects of the mutations on the detection range or output were confirmed in the BmoR mutants. Four combinatorial mutants containing one increased-detection-range mutation and one enhanced-output mutation were constructed. Compared with wild-type BmoR, F276A/E627N BmoR and D333N/E627N BmoR have wider detection ranges (0-100 mM) and relatively high outputs to the isobutanol added quantitatively or produced intracellularly, demonstrating they have potential for screening isobutanol overproduction strains. This work presented an example of engineering the wild-type transcription factors with physiological significance for industrial utilization.


Assuntos
Proteínas de Bactérias/química , Butanóis/química , Mutação de Sentido Incorreto , Fatores de Transcrição/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Butanóis/metabolismo , Mutagênese Sítio-Dirigida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
J Vis Exp ; (148)2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31282885

RESUMO

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.


Assuntos
Aminoácidos/biossíntese , Códon/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Marcadores Genéticos , Mutação/genética , Plasmídeos/genética , Biossíntese de Proteínas , RNA de Transferência/metabolismo
16.
J Biotechnol ; 301: 97-104, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31181238

RESUMO

Protein purification is an indispensable step in diverse fields of biological research or production process. Conventional purification methods including the affinity purification or the usage of self-aggregating tags suffered from many drawbacks such as the complicated steps, high cost and low efficiency. Moreover, the fusion tag usually had negative effects on the activity of the target protein. To address the above issues, here we propose a novel protein purification method which needs simple operation steps, and this method is mediated by the combination of CipA protein and a mini-intein (Synechocystis sp. PCC6803 DnaB, Ssp DnaB), depending on the assembly function of CipA and the self-cleavage function of Ssp DnaB. To realize the purification, CipA-DnaB-eGFP protein was expressed and assembled into protein crystalline inclusions (PCIs) in E. coli. Then, only cell lysis, cleavage and centrifugation steps were required to purify eGFP. Purified eGFP was in the supernatant with a purity of over 90%. The cleavage efficiency and the yield of eGFP reached 51.96% and 13.99 ±â€¯0.88 mg/L fermentation broth, respectively. Furthermore, to broaden the application of this approach, three other proteins which were maltose binding protein (MBP), ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhP) were purified with high cleavage efficiency. The purified Kivd and AdhP remained high specific activities. This work demonstrated an effective and convenient protein purification method.


Assuntos
Proteínas de Bactérias/genética , Inteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Synechocystis/genética , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
17.
Microb Cell Fact ; 18(1): 60, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909908

RESUMO

BACKGROUND: Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specific and programmable, with efficiencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR-Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efficiency is currently less than 15%, due to false positives caused by the poor targeting efficiency of the crRNA. RESULTS: To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efficiency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR-Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efficient C. glutamicum crRNA contained a 5'-NYTV-3' PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks. CONCLUSIONS: Here, we identified optimized PAM-related parameters for the CRISPR-Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1-based genome editing. This optimized system, with higher editing efficiency, could be used to increase the production of bulk chemicals, such as isobutyrate, in C. glutamicum.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , RNA/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Endonucleases/fisiologia , Edição de Genes
18.
Microb Cell Fact ; 18(1): 30, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732651

RESUMO

BACKGROUND: Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4-C6 linear alcohols and C3-C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library. RESULTS: Firstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment. CONCLUSIONS: This work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.


Assuntos
Técnicas Biossensoriais , Butanóis/metabolismo , Engenharia Metabólica/métodos , Vias Biossintéticas , Butanóis/análise , Fermentação , Microbiologia Industrial , Mutagênese , Mutação , Thauera/genética , Thauera/metabolismo
19.
Nat Commun ; 9(1): 3616, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190534

RESUMO

The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation. Theoretically, disrupted or retarded translation caused by the lack of charged rare tRNAs can be partially restored by feeding or intracellular synthesis of the corresponding amino acids. Inspired by this assumption, we develop a screening or selection system for obtaining overproducers of a target amino acid by replacing its common codons with the corresponding synonymous rare alternative in the coding sequence of selected reporter proteins or antibiotic-resistant markers. Results show that integration of rare codons can inhibit gene translations in a frequency-dependent manner. As a proof-of-concept, Escherichia coli strains overproducing L-leucine, L-arginine or L-serine are successfully selected from random mutation libraries. The system is also applied to Corynebacterium glutamicum to screen out L-arginine overproducers. This strategy sheds new light on obtaining and understanding amino acid overproduction strains.


Assuntos
Aminoácidos/biossíntese , Códon , Corynebacterium glutamicum/genética , Escherichia coli/genética , Engenharia Genética/métodos , Aminoácidos/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Microrganismos Geneticamente Modificados
20.
Appl Microbiol Biotechnol ; 102(23): 10005-10015, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30242435

RESUMO

Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.


Assuntos
Carboxiliases/metabolismo , Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Pirogalol/metabolismo , Carbono/metabolismo , Meios de Cultura/química , DNA Bacteriano/genética , Escherichia coli/genética , Ácido Gálico/metabolismo , Hidroxibenzoatos/metabolismo , Corpos de Inclusão/metabolismo , Indóis/metabolismo , Microbiologia Industrial , Klebsiella pneumoniae/enzimologia , Propionatos/metabolismo , Pseudomonas aeruginosa/enzimologia
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