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1.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
2.
Appl Microbiol Biotechnol ; 103(5): 2087-2099, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30661108

RESUMO

Bioethanol has been considered as a potentially renewable energy source, and metabolic engineering plays an important role in the production of biofuels. As an efficient ethanol-producing bacterium, Zymomonas mobilis has garnered special attention due to its high sugar uptake, ethanol yield, and tolerance. Different metabolic engineering strategies have been used to establish new metabolic pathways for Z. mobilis to broaden its substrate range, remove competing pathways, and enhance its tolerance to ethanol and lignocellulosic hydrolysate inhibitors. Recent advances in omics technology, computational modeling and simulation, system biology, and synthetic biology contribute to the efficient re-design and manipulation of microbes via metabolic engineering at the whole-cell level. In this review, we summarize the progress of some new technologies used for metabolic engineering to improve bioethanol production and tolerance in Z. mobilis. Some successful examples of metabolic engineering used to develop strains for ethanol production are described in detail. Lastly, some important strategies for future metabolic engineering efforts are also highlighted.


Assuntos
Biocombustíveis/microbiologia , Etanol/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Zymomonas/metabolismo , Farmacorresistência Bacteriana/genética , Fermentação , Redes e Vias Metabólicas/genética , Zymomonas/genética
3.
Nat Protoc ; 14(2): 415-440, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30635653

RESUMO

The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical-genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.


Assuntos
Interação Gene-Ambiente , Genoma Bacteriano , Genoma Fúngico , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Software , Código de Barras de DNA Taxonômico/métodos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/classificação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Zymomonas/classificação , Zymomonas/efeitos dos fármacos , Zymomonas/genética , Zymomonas/metabolismo
4.
J Biotechnol ; 282: 32-37, 2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-29807049

RESUMO

Inhibition of sodium ion (Na+) on Zymomonas mobilis represents an important obstacle for efficient cellulosic ethanol production. This study screened and overexpressed the genes responsible for transporting metal ions in Z. mobilis for increasing its Na+ tolerance. The ZMO0119 gene encoding Na+/H+ antiporter was identified to be highly effective for reducing intracellular Na+ concentration of Z. mobilis by improving the Na+ transport capacity. Overexpression of ZMO0119 gene in Z. mobilis significantly accelerated the cell growth, glucose consumption, and cellulosic ethanol production from the dry acid pretreated and biodetoxified corn stover feedstock. This study provided an important gene responsible for increasing the cellulosic ethanol fermentability by Z. mobilis.


Assuntos
Fermentação/genética , Genes Bacterianos , Tolerância ao Sal/genética , Zymomonas/genética , Zymomonas/metabolismo , Celulose/metabolismo , Etanol/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Sódio , Zea mays , Zymomonas/crescimento & desenvolvimento
5.
Lett Appl Microbiol ; 67(1): 54-63, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29603295

RESUMO

Zymomonas mobilis has long attracted attention owing to its capacity to ferment hexose to ethanol. From a taxonomic viewpoint, Z. mobilis is a unique species of the genus Zymomonas, separated into three subspecies, Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis subsp. francensis on the basis of physiological tests, which are often unreliable owing to the genetic proximity among these species. Currently, the use of molecular techniques is more appropriate for identification of these bacterial subspecies. In this study, the 32 strains of Z. mobilis present in the UFPEDA bacterial collection were characterized using molecular techniques, such as sequencing of the 16S rDNA gene and its theoretical restriction profile, classifying them as members of the subspecies, Z. mobilis subsp. mobilis. In addition, anaerobic cultivations were performed, which showed the biological diversity of the strains in terms of growth, sugar consumption and ethanol production. From these results, it was possible to identify the strain Z-2-80 as a promising bacterium for use in the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Zymomonas mobilis is a bacterium of great relevance to biotechnology, owing to its capacity to ferment hexose to ethanol. On a molecular basis, 32 isolates were identified as Z. mobilis subsp. mobilis. However, intraspecific diversity was identified when these were grown under strictly anaerobic conditions. The results obtained from this study suggest a strain of Z. mobilis as an alternative for use in the fermentation process.


Assuntos
Reatores Biológicos/microbiologia , DNA Bacteriano/genética , Etanol/metabolismo , Zymomonas/classificação , Zymomonas/metabolismo , Anaerobiose , Brasil , DNA Ribossômico/genética , Fermentação , Hexoses/metabolismo , RNA Ribossômico 16S/genética , Zymomonas/genética , Zymomonas/isolamento & purificação
6.
PLoS One ; 13(4): e0195994, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29694430

RESUMO

Zymomonas mobilis has the special Entner-Doudoroff (ED) pathway and it has excellent industrial characteristics, including low cell mass formation, high-specific productivity,ethanol yield, notable ethanol tolerance and wide pH range, a relatively small genome size. In this study, the genome sequences of NRRL B-14023 and NRRL B-12526 were sequenced and compared with other strains to explore their evolutionary relationships and the genetic basis of Z. mobilis. The comparative genomic analyses revealed that the 8 strains share a conserved core chromosomal backbone. ZM4, NRRL B-12526, NRRL B-14023, NCIMB 11163 and NRRL B-1960 share 98% sequence identity across the whole genome sequences. Highly similar plasmids and CRISPR repeats were detected in these strains. A whole-genome phylogenetic tree of the 8 strains indicated that NRRL B-12526, NRRL B-14023 and ATCC 10988 had a close evolutionary relationship with the strain ZM4. Furthermore, strains ATCC29191 and ATCC29192 had distinctive CRISPR with a far distant relationship. The size of the pan-genome was 1945 genes, including 1428 core genes and 517 accessory genes. The genomes of Z. mobilis were highly conserved; particularly strains ZM4, NRRL B-12526, NRRL B-14023, NCIMB 11163 and NRRL B-1960 had a close genomic relationship. This comparative study of Z. mobilis presents a foundation for future functional analyses and applications.


Assuntos
Genoma Bacteriano , Análise de Sequência de DNA/métodos , Zymomonas/classificação , Etanol/metabolismo , Evolução Molecular , Tamanho do Genoma , Concentração de Íons de Hidrogênio , Filogenia , Zymomonas/genética , Zymomonas/metabolismo
7.
Appl Microbiol Biotechnol ; 102(7): 3337-3347, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29464326

RESUMO

Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.


Assuntos
Biocombustíveis/microbiologia , Farmacorresistência Bacteriana/genética , Furaldeído/farmacologia , Reação em Cadeia da Polimerase , Zymomonas/efeitos dos fármacos , Zymomonas/genética , Embaralhamento de DNA , Mutação
8.
Electron. j. biotechnol ; 30: 118-124, nov. 2017. tab, ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1021652

RESUMO

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.


Assuntos
Zymomonas/genética , Recombinação Homóloga , Plasmídeos , Recombinação Genética , Álcool Desidrogenase/metabolismo , Zymomonas/enzimologia , Eletroporação , Etanol/metabolismo , Técnicas de Inativação de Genes , Mutação
9.
Microb Cell Fact ; 16(1): 171, 2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28978312

RESUMO

BACKGROUND: Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. RESULT: Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. CONCLUSION: The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield.


Assuntos
Clostridium thermocellum/enzimologia , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Piruvato Descarboxilase/metabolismo , Acetobacteraceae/enzimologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Celulose/metabolismo , Clostridium thermocellum/genética , Fermentação , Engenharia Metabólica , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/isolamento & purificação , Temperatura Ambiente , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo , Zymomonas/genética , Zymomonas/metabolismo
10.
BMC Biotechnol ; 17(1): 63, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28764759

RESUMO

BACKGROUND: Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. RESULTS: In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. CONCLUSION: The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.


Assuntos
Ácido Acético/farmacologia , Etanol/metabolismo , Zymomonas/efeitos dos fármacos , Zymomonas/metabolismo , Ácido Acético/metabolismo , Fermentação , Engenharia Genética/métodos , Metilnitronitrosoguanidina/farmacologia , Mutagênese , Mutação , Zymomonas/genética
11.
FEMS Microbiol Lett ; 364(13)2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28655181

RESUMO

The bacterium Zymomonas mobilis naturally produces ethanol at near theoretical maximum yields, making it of interest for industrial ethanol production. Zymomonas mobilis requires the vitamin pantothenate for growth. Here we characterized the genetic basis for the Z. mobilis pantothenate auxotrophy. We found that this auxotrophy is due to the absence of a single gene, panD, encoding aspartate-decarboxylase. Heterologous expression of Escherichia coli PanD in Z. mobilis or supplementation of the growth medium with the product of PanD activity, ß-alanine, eliminated the need for exogenous pantothenate. We also determined that Z. mobilis IlvC, an enzyme better known for branched-chain amino acid synthesis, is required for pantothenate synthesis in Z. mobilis, as it compensates for the absence of PanE, another pantothenate synthesis pathway enzyme. In addition to contributing to an understanding of the nutritional requirements of Z. mobilis, our results have led to the design of a more cost-effective growth medium.


Assuntos
Carboxiliases/metabolismo , Etanol/metabolismo , Ácido Pantotênico/deficiência , Zymomonas/enzimologia , Zymomonas/crescimento & desenvolvimento , Aminoácidos de Cadeia Ramificada/biossíntese , Aminoácidos de Cadeia Ramificada/genética , Carboxiliases/genética , Meios de Cultura/economia , Meios de Cultura/metabolismo , Proteínas de Escherichia coli/genética , Fermentação , Expressão Gênica , Vetores Genéticos/genética , Ácido Pantotênico/genética , Zymomonas/genética , beta-Alanina/metabolismo
12.
Appl Microbiol Biotechnol ; 101(12): 5089-5099, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28341886

RESUMO

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l-1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l-1) together with sorbitol (65 g l-1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l-1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.


Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Gluconatos/metabolismo , Microbiologia Industrial , Lactonas/metabolismo , Zymomonas/enzimologia , Zymomonas/fisiologia , Biomassa , Biotransformação , Etanol/metabolismo , Fermentação , Frutose/metabolismo , Técnicas de Inativação de Genes , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Sorbitol/metabolismo , Estresse Fisiológico , Zymomonas/genética , Zymomonas/crescimento & desenvolvimento
13.
Biosci Biotechnol Biochem ; 81(3): 453-459, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27900888

RESUMO

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Inativação de Genes/métodos , Plasmídeos/genética , Zymomonas/genética , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Endonucleases/genética , Escherichia coli/genética , Dosagem de Genes , Zymomonas/crescimento & desenvolvimento
14.
Proteins ; 85(2): 312-321, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27936490

RESUMO

It is important to identify hotspot residues that determine protein-protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA-HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA-HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site-directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high-affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. Proteins 2017; 85:312-321. © 2016 Wiley Periodicals, Inc.


Assuntos
Aminoidrolases/química , Subunidades Proteicas/química , Pyrobaculum/genética , Thermotoga maritima/genética , Zymomonas/genética , Aminoidrolases/genética , Aminoidrolases/metabolismo , Sítios de Ligação , Evolução Biológica , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Filogenia , Ligação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pyrobaculum/classificação , Pyrobaculum/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Thermotoga maritima/classificação , Thermotoga maritima/enzimologia , Zymomonas/classificação , Zymomonas/enzimologia
15.
World J Microbiol Biotechnol ; 32(12): 194, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27722827

RESUMO

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.


Assuntos
Enterobacter/genética , Fosfatos/química , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo , Enterobacter/metabolismo , Engenharia Genética , Gluconatos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Zymomonas/enzimologia , Zymomonas/genética , beta-Frutofuranosidase/genética
16.
Microb Biotechnol ; 9(6): 699-717, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27629544

RESUMO

Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. This review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.


Assuntos
Biocombustíveis , Produtos Biológicos/metabolismo , Zymomonas/metabolismo , Biotecnologia/métodos , Fermentação , Engenharia Metabólica/métodos , Zymomonas/genética
17.
ACS Synth Biol ; 5(12): 1519-1534, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27404024

RESUMO

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosome-binding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe2+-dependent AdhB and Zn2+-dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Engenharia Metabólica/métodos , Zymomonas/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Óperon , Regiões Promotoras Genéticas , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Zinco/metabolismo , Zymomonas/genética , Zymomonas/crescimento & desenvolvimento
18.
Microb Cell Fact ; 15(1): 101, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27287016

RESUMO

BACKGROUND: The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. RESULTS: Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. CONCLUSIONS: The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.


Assuntos
Tolerância ao Sal/genética , Transposases/genética , Zymomonas/genética , Zymomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Engenharia Genética , Glucose/metabolismo , Mutagênese Insercional , NAD/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Transposases/metabolismo , Zymomonas/crescimento & desenvolvimento
19.
J Ind Microbiol Biotechnol ; 43(6): 861-71, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27033536

RESUMO

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.


Assuntos
Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Ácidos Graxos/biossíntese , Zymomonas/metabolismo , Aminoácidos/química , Proteínas de Bactérias/genética , Biomassa , Meios de Cultura/química , DNA Bacteriano/genética , Fermentação , Glucose/química , Temperatura Alta , Microbiologia Industrial , Engenharia Metabólica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitaminas/química , Xilose/química , Zymomonas/genética
20.
Mol Biosyst ; 12(4): 1241-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26883123

RESUMO

Zymomonas mobilis is an ethanologenic bacterium and is known to be an example microorganism with energy-uncoupled growth. A genome-scale metabolic model could be applicable for understanding the characteristics of Z. mobilis with rapid catabolism and inefficient energy conversion. In this study, a charge balanced genome-scale metabolic model (iEM439) of Z. mobilis ATCC 10988 (ZM1) including 439 genes, 692 metabolic reactions and 658 metabolites was reconstructed based on genome annotation and previously published information. The model presents a much better prediction for biomass and ethanol concentrations in a batch culture by using dynamic flux balance analysis compared with the two previous genome-scale metabolic models. Furthermore, intracellular flux distribution obtained from the model was consistent with the fluxes for glucose fermentation determined by (13)C NMR. The model predicts that there is no difference in growth rates of Z. mobilis under aerobic and anaerobic conditions whereas ethanol production is decreased and production of other metabolites including acetate and acetoin is increased under aerobic conditions. Experimental data confirm the predicted differences between the aerobic and anaerobic growth of Z. mobilis. Finally, the model was used to study the energy-uncoupled growth of Z. mobilis and to predict its effect on flux distribution in the central metabolism. Flux distribution obtained from the model indicates that coupling growth and energy reduces ethanol secretion and changes the flux distribution to produce more biomass. This coupling is also associated with a significant increase in the proton uptake rate based on the prediction of the charge balanced model. Hence, resistance to intracellular pH reduction could be the main reason for uncoupled growth and Z. mobilis uses ATPase to pump out the proton. Experimental observations are in accordance with the predicted relationship between growth, ATP dissipation and proton exchange.


Assuntos
Estudo de Associação Genômica Ampla , Metaboloma , Metabolômica , Modelos Biológicos , Zymomonas/genética , Zymomonas/metabolismo , Adenosina Trifosfatases/metabolismo , Algoritmos , Simulação por Computador , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Metabolômica/métodos , Consumo de Oxigênio , ATPases Translocadoras de Prótons/metabolismo , Reprodutibilidade dos Testes
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