scholarly journals Improvement of CO2 and Acetate Coupling into Lactic Acid by Genetic Manipulation of the Hyperthermophilic Bacterium Thermotoga neapolitana

2021 ◽  
Vol 9 (8) ◽  
pp. 1688
Author(s):  
Nunzia Esercizio ◽  
Mariamichela Lanzilli ◽  
Marco Vastano ◽  
Zhaohui Xu ◽  
Simone Landi ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Carbon Capture ◽  
Recombinant Strain ◽  
Genetic Manipulation ◽  
Lactate Production ◽  
Value Added ◽  
Thermotoga Neapolitana ◽  
Hydrogen Yield ◽  
Wild Type ◽  
Future Studies

Capnophilic lactic fermentation (CLF) represents an attractive biotechnological process for biohydrogen production and synthesis of L-lactic acid from acetate and CO2. The present study focuses on a genetic manipulation approach of the Thermotoga neapolitana DSM33003 strain to enhance lactic acid synthesis by the heterologous expression of a thermostable acetyl-CoA synthetase that catalyses the irreversible acetate assimilation. Because of the scarcity of available genetic tools, each transformation step was optimized for T. neapolitana DSM33003 to cope with the specific needs of the host strain. Batch fermentations with and without an external source of acetate revealed a strongly increased lactate production (up to 2.5 g/L) for the recombinant strain compared to wild type. In the engineered bacterium, the assimilation of CO2 into lactic acid was increased 1.7 times but the hydrogen yield was impaired in comparison to the wild type strain. Analysis of fermentation yields revealed an impaired metabolism of hydrogen in the recombinant strain that should be addressed in future studies. These results offer an important prospective for the development of a sustainable approach that combines carbon capture, energy production from renewable source, and the synthesis of high value-added products, which will be addressed in future studies.

scholarly journals Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

2012 ◽  
Vol 78 (19) ◽  
pp. 7098-7106 ◽  
Author(s):  
S. Andreas Angermayr ◽  
Michal Paszota ◽  
Klaas J. Hellingwerf
Keyword(s):  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Lactic Acid Production ◽  
Lactate Production ◽  
Cell Factory ◽  
Wild Type ◽  
Extracellular Medium ◽  
Content Type ◽  
Bulk Chemicals ◽  
Versatile Tool

ABSTRACTMetabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO2has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of anldhgene fromBacillus subtilis(encoding anl-lactate dehydrogenase) into the genome ofSynechocystissp. strain PCC6803 leads tol-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the singleldhmutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

scholarly journals Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

2004 ◽  
Vol 70 (8) ◽  
pp. 4596-4603 ◽  
Author(s):  
Katy Vaillancourt ◽  
Jean-Dominique LeMay ◽  
Maryse Lamoureux ◽  
Michel Frenette ◽  
Sylvain Moineau ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Generation Time ◽  
Recombinant Strain ◽  
Wild Type Strain ◽  
External Medium ◽  
Streptococcus Thermophilus ◽  
Mozzarella Cheese ◽  
Wild Type ◽  
Leloir Pathway

ABSTRACT The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal− strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal+ strain grew and produced acid more rapidly than the Gal− wild-type strain.

scholarly journals Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Jong Kwan Lee ◽  
Sujin Kim ◽  
Wonsik Kim ◽  
Sungil Kim ◽  
Seungwoo Cha ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Value Added ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Gene Encoding ◽  
Laboratory Evolution ◽  
Tolerant Strain ◽  
Dehydrogenase Gene

Abstract Background Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. Results In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. Conclusions JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

scholarly journals Breeder Diet Strategies for Generating Ttpa-Null and Wild-Type Mice with Low Vitamin E Status to Assess Neurological Outcomes

2020 ◽  
Vol 4 (11) ◽  
Author(s):  
Katherine M Ranard ◽  
Matthew J Kuchan ◽  
John W Erdman
Keyword(s):  
Animal Model ◽  
Vitamin E ◽  
Mouse Model ◽  
Wild Type ◽  
Future Studies ◽  
Neurological Outcomes ◽  
Transfer Protein ◽  
And Function ◽  
The Brain ◽  
Vitamin E Status

ABSTRACT Studying vitamin E [α-tocopherol (α-T)] metabolism and function in the brain and other tissues requires an animal model with low α-T status, such as the transgenic α-T transfer protein (Ttpa)–null (Ttpa−/−) mouse model. Ttpa+/− dams can be used to produce Ttpa−/− and Ttpa+/+mice for these studies. However, the α-T content in Ttpa+/− dams’ diet requires optimization; diets must provide sufficient α-T for reproduction, while minimizing the transfer of α-T to the offspring destined for future studies that require low baseline α-T status. The goal of this work was to assess the effectiveness and feasibility of 2 breeding diet strategies on reproduction outcomes and offspring brain α-T concentrations. These findings will help standardize the breeding methodology used to generate the Ttpa−/− mice for neurological studies.

scholarly journals Insights into neutralizing antibody responses in individuals exposed to SARS-CoV-2 in Chile

2021 ◽  
Vol 7 (7) ◽  
pp. eabe6855 ◽  
Author(s):  
Carolina Beltrán-Pavez ◽  
Sebastián Riquelme-Barrios ◽  
Aarón Oyarzún-Arrau ◽  
Aracelly Gaete-Argel ◽  
Roxana González-Stegmaier ◽  
...  
Keyword(s):  
General Population ◽  
Local Level ◽  
Neutralizing Antibody ◽  
Host Immunity ◽  
Antibody Responses ◽  
Wild Type ◽  
Neutralization Activity ◽  
Future Studies

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19–related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.

scholarly journals Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yu Gao ◽  
Yan Sun ◽  
Huiling Gao ◽  
Ying Chen ◽  
Xiaoqing Wang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Negative Impact ◽  
Value Added ◽  
Cyperus Esculentus ◽  
Wild Type ◽  
Tobacco Leaves ◽  
Vegetative Tissues ◽  
Oil Storage ◽  
Tag Biosynthesis

Abstract Background Engineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops. Results Three CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content. Conclusions The present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

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