scholarly journals Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247135
Author(s):  
Dung Minh Ha-Tran ◽  
Rou-Yin Lai ◽  
Trinh Thi My Nguyen ◽  
Eugene Huang ◽  
Shou-Chen Lo ◽  
...  
Keyword(s):  
Rice Straw ◽  
Kluyveromyces Marxianus ◽  
Rhodopseudomonas Palustris ◽  
Napier Grass ◽  
Purple Bacterium ◽  
Growth Media ◽  
Bisphosphate Carboxylase ◽  
Hexose Sugars ◽  
Microbial System ◽  
Pentose Sugars

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Effects of Protein and Roughage Sources in Total Mixed Ration on Voluntary Feed Intake, Nutrient Intake and Blood Metabolites in Black Bengal Goats

2021 ◽  
Author(s):  
W. Polviset ◽  
N. Danopas
Keyword(s):  
Rice Straw ◽  
Feed Intake ◽  
Nutrient Intake ◽  
Agricultural Productivity ◽  
Latin Square ◽  
Neutral Detergent Fiber ◽  
Napier Grass ◽  
Blood Metabolites ◽  
Cassava Leaves ◽  
Voluntary Feed Intake

Background: Agricultural productivity in Southeast Asian countries are important to produce food for human, thus the first priority to improve agricultural productivity is feed and feeding in livestock, especially in ruminants when rice straw and Napier grass are used as the roughage sources; in addition, cassava leaves and Leucaena leaves can be used as the protein sources. Interestingly, the strategy to improve feed in ruminant is through the use of total mixed rations (TMR) which are produced by combination of roughages, concentrates, minerals, vitamins and additives. Methods: During the period 2020-2021 a study with 2 × 2 factorial, in 4 × 4 Latin square design with 21 days per period tested the following TMR’s T1- 5% urea treated rice straw with dried Leucaena leaves, T2- 5% urea treated rice with dried cassava leaves, T3- fermented napier grass with dried Leucaena leaves and the T4- fermented napier grass with dried cassava leaves on voluntary feed intake, nutrient intake and blood metabolites in Black Bengal goats. Result: All treatments did not affect voluntary feed intake (kgDM/head/day and % BW) (P greater than 0.05), but feeding with 5% urea treated rice with dried cassava leaves, it was non significantly higher (0.71 kgDM/day). Furthermore, nutrient intake of organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and rumination were not affected among all the four treatments (P greater than 0.05). Additionally, blood glucose, blood urea nitrogen and triglyceride concentrations in plasma were not influenced due to treatments (P greater than 0.05). Nevertheless, Feed cost (USD/kg) was reduced T3 and T4. In conclusion, feeding Black Bengal goat with fermented napier grass with dried Leucaena leaves and fermented napier grass with dried cassava leaves in the TMR were suitable because of the lowest price (0.13 USD/kg) when compared to the other treatments.

scholarly journals Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris

2005 ◽  
Vol 187 (22) ◽  
pp. 7784-7794 ◽  
Author(s):  
Yasuhiro Oda ◽  
Sudip K. Samanta ◽  
Federico E. Rey ◽  
Liyou Wu ◽  
Xiudan Liu ◽  
...  
Keyword(s):  
Nitrogen Availability ◽  
Rhodopseudomonas Palustris ◽  
Genomic Analysis ◽  
Photosynthetic Bacterium ◽  
Growth Media ◽  
Nitrogen Fixing ◽  
Nitrogen Acquisition ◽  
Functional Genomic Analysis ◽  
Alternative Nitrogenase ◽  
Dependent Growth

ABSTRACT The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing medium. Strains that were unable to express a functional Mo nitrogenase due to mutations in Mo nitrogenase structural genes synthesized functional V and Fe nitrogenases and expressed vnf and anf genes in nitrogen-fixing growth media that contained Mo and V at concentrations far in excess of those that repress alternative nitrogenase gene expression in other bacteria. Thus, not only does R. palustris have multiple enzymatic options for nitrogen fixation, but in contrast to reports on other nitrogen-fixing bacteria, the expression of its alternative nitrogenases is not repressed by transition metals. Between 95 and 295 genes that are not directly associated with nitrogenase synthesis and assembly were induced under nitrogen-fixing conditions, depending on which nitrogenase was being used by R. palustris. Genes for nitrogen acquisition were expressed at particularly high levels during alternative nitrogenase-dependent growth. This suggests that alternative nitrogenase-expressing cells are relatively starved for nitrogen and raises the possibility that fixed nitrogen availability may be the primary signal that controls the synthesis of the V and Fe nitrogenases.

scholarly journals A Novel Three-Protein Two-Component System Provides a Regulatory Twist on an Established Circuit To Modulate Expression of the cbbI Region of Rhodopseudomonas palustris CGA010

2006 ◽  
Vol 188 (8) ◽  
pp. 2780-2791 ◽  
Author(s):  
Simona Romagnoli ◽  
F. Robert Tabita
Keyword(s):  
Response Regulator ◽  
Rhodopseudomonas Palustris ◽  
Sensor Kinase ◽  
Response Regulators ◽  
Transcription Activator ◽  
Two Component System ◽  
Bisphosphate Carboxylase ◽  
Control Of Gene Expression ◽  
Component System ◽  
Two Component

ABSTRACT A novel two-component system has been identified in the cbbI region of the nonsulfur purple photosynthetic bacterium Rhodopseudomonas palustris. Genes encoding this system, here designated cbbRRS, are juxtaposed between the divergently transcribed transcription activator gene, cbbR, and the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS. The three genes of the cbbRRS system represent a variation of the well-known two-component signal transduction systems, as there are a transmembrane hybrid sensor kinase and two response regulators, with no apparent DNA binding domain associated with any of the three proteins encoded by these genes. In this study, we showed that the membrane-bound full-length kinase undergoes autophosphorylation and transfers phosphate to both response regulators. A soluble, truncated version of the kinase was subsequently prepared and found to catalyze phosphorylation of response regulator 1 but not response regulator 2, implying that conformational changes and/or sequence-specific regions of the kinase are important for discriminating between the two response regulators. Analyses indicated that a complex network of control of gene expression must occur, with CbbR required for the expression of the cbbLS genes but dispensable for the synthesis of form II RubisCO (encoded by cbbM). The CbbRRS proteins specifically affected the activity and accumulation of form I RubisCO (CbbLS), as revealed by analyses of nonpolar, unmarked gene deletions. A tentative model of regulation suggested that changes in the phosphotransfer activity of the sensor kinase, possibly in response to a redox metabolic signal, cause modulation of the activity and synthesis of form I RubisCO.

Degradation of Lignocelluloses in Rice Straw by BMC-9, a Composite Microbial System

2014 ◽  
Vol 24 (5) ◽  
pp. 585-591 ◽  
Author(s):  
Hongyan Zhao ◽  
Hairu Yu ◽  
Xufeng Yuan ◽  
Renzhe Piao ◽  
Hulin Li ◽  
...  
Keyword(s):  
Rice Straw ◽  
Microbial System

scholarly journals Differential Accumulation of Form I RubisCO in Rhodopseudomonas palustris CGA010 under Photoheterotrophic Growth Conditions with Reduced Carbon Sources

2009 ◽  
Vol 191 (13) ◽  
pp. 4243-4250 ◽  
Author(s):  
Gauri S. Joshi ◽  
Simona Romagnoli ◽  
Nathan C. VerBerkmoes ◽  
Robert L. Hettich ◽  
Dale Pelletier ◽  
...  
Keyword(s):  
Purple Bacteria ◽  
Rhodopseudomonas Palustris ◽  
Growth Conditions ◽  
Activity Levels ◽  
Two Component System ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Component System ◽  
Two Component ◽  
Photoheterotrophic Growth

ABSTRACT Rhodopseudomonas palustris is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO2 or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO2 in the medium to support growth. These proteins were not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene, cbbR, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the cbbRRS genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence cbb transcription.

scholarly journals Significant Role for Microbial Autotrophy in the Sequestration of Soil Carbon

2012 ◽  
Vol 78 (7) ◽  
pp. 2328-2336 ◽  
Author(s):  
Hongzhao Yuan ◽  
Tida Ge ◽  
Caiyan Chen ◽  
Anthony G. O'Donnell ◽  
Jinshui Wu
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Fragment Length Polymorphism ◽  
Ralstonia Eutropha ◽  
Gene Diversity ◽  
Rhodopseudomonas Palustris ◽  
C Sequestration ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Content Type ◽  
Autotrophic Microorganisms

ABSTRACTSoils were incubated for 80 days in a continuously labeled14CO2atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of14C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 108km2, this represents a potential global sequestration of 0.6 to 4.9 Pg C year−1. Estimated global C sequestration rates suggest a “missing sink” for carbon of between 2 and 3 Pg C year−1. To determine whether14CO2incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO2-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominantcbbL-containing bacteria wereAzospirillum lipoferum,Rhodopseudomonas palustris,Bradyrhizobium japonicum,Ralstonia eutropha, andcbbL-containing chromophytic algae of the generaXanthophytaandBacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences incbbL-containing microbial communities between soils. Differences incbbLgene diversity were shown to be correlated with differences in SOC content. Bacterial and algalcbbLgene abundances were between 106and 108and 103to 105copies g−1soil, respectively. BacterialcbbLabundance was shown to be positively correlated with RubisCO activity (r= 0.853;P< 0.05), and bothcbbLabundance and RubisCO activity were significantly related to the synthesis rates of [14C]SOC (r= 0.967 and 0.946, respectively;P< 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.

scholarly journals Genome Sequence of the Chemolithoautotrophic Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Nb-255

2006 ◽  
Vol 72 (3) ◽  
pp. 2050-2063 ◽  
Author(s):  
Shawn R. Starkenburg ◽  
Patrick S. G. Chain ◽  
Luis A. Sayavedra-Soto ◽  
Loren Hauser ◽  
Miriam L. Land ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Rhodopseudomonas Palustris ◽  
Type I ◽  
Nitrite Oxidation ◽  
Circular Chromosome ◽  
Bisphosphate Carboxylase ◽  
Gene Copies ◽  
Nitrite Oxidizing Bacteria ◽  
Genes Encoding ◽  
Nitrite Oxidoreductase

ABSTRACT The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.

scholarly journals Engineering of a Type III Rubisco from a Hyperthermophilic Archaeon in Order To Enhance Catalytic Performance in Mesophilic Host Cells

2007 ◽  
Vol 73 (19) ◽  
pp. 6254-6261 ◽  
Author(s):  
Shosuke Yoshida ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Rhodopseudomonas Palustris ◽  
Host Cells ◽  
Mutant Protein ◽  
Wild Type ◽  
Turnover Number ◽  
Bisphosphate Carboxylase ◽  
Mutant Proteins ◽  
Hyperthermophilic Archaeon ◽  
Type Iii ◽  
Α Helix

ABSTRACT The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (RbcTk). It has previously been shown that RbcTk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Δ3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of RbcTk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and α-helix 6 regions among RbcTk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of RbcTk is one residue longer than those found in the spinach and G. partita enzymes, and replacing RbcTk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Δ3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type RbcTk. SP6 corresponds to a complete substitution of the original α-helix 6 of RbcTk with that of the spinach enzyme. Compared to wild-type RbcTk, the purified SP6 mutant protein exhibited a 30% increase in turnover number (k cat) of the carboxylase activity and a 17% increase in the k cat/Km value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in RbcTk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of RbcTk at ambient temperatures.

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