Using Genome-Wide Protein Sequence Data to Predict Amino Acid Conservation

For most proteins, multiple sequence alignments are a viable method to identify functionally and structurally important amino acids, but for most organisms, there is a subset of proteins that are unique or found in a few closely related organisms. For these proteins, it is not possible to produce sequence alignments that are useful in identifying functionally or structurally important amino acids. We have investigated the relationship between amino acid conservation and five factors (the amino acid’s identity, N-terminal neighbor, C-terminal neighbor, the local hydropathy of surrounding amino acids, and the local expected net charge of the surrounding amino acids based on the primary sequence) in Escherichia coli proteins. For four of the factors examined (all but the amino acid’s identity), there is a significant relationship with conservation for some of the standard 20 amino acids. Using the combination of all five factors, we show that it is possible to calculate a score based on the primary sequences of a subset of E. coli proteins that has statistically significant predictive value with respect to predicting conserved amino acids in other E. coli proteins and Saccharomyces cerevisiae proteins. As these five variables show significant relationships with conservation, we have termed them conservation factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of amino acid supplementation on the synthesis of poly(3-hydroxybutyrate) by recombinant pha Sa + Escherichia coli

The effect of different amino acid supplements to the basal medium on poly(3-hydroxybutyrate) (PHB) accumulation by recombinant pha _Sa ⁺ Escherichia coli (ATCC: PTA-1579) harbouring the poly(3-hydroxybutyrate)-synthesizing genes from Streptomyces aureofaciens NRRL 2209 was studied. With the exception of glycine and valine, all other amino acid supplements brought about enhancement of PHB accumulation. In particular, cysteine, isoleucine or methionine supplementation increased PHB accumulation by 60, 45 and 61% respectively by the recombinant E. coli as compared with PHB accumulation by this organism in the basal medium. The effect of co-ordinated addition of assorted combinations of these three amino acids on PHB accumulation was studied using a 2³ factorial design. The three-factor interaction analyses revealed that the effect of the three amino acids on PHB accumulation by the recombinant E. coli was in the order of cysteine > methionine > isoleucine. The defined medium supplemented with cysteine, methionine and isoleucine at the concentration of 150 mgl^–1 each and glycerol as the carbon source was the optimum medium that resulted in the accumulation of about 52% PHB of cell dry weight.     

An economical method for producing stable-isotope labeled proteins by the <Emphasis Type="Italic">E. coli</Emphasis> cell-free system

Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.     

Selection on Synthesis Cost Affects Interprotein Amino Acid Usage in All Three Domains of Life

Most investigations of the forces shaping protein evolution have focussed on protein function. However, cells are typically 50%–75% protein by dry weight, with protein expression levels distributed over five orders of magnitude. Cells may, therefore, be under considerable selection pressure to incorporate amino acids that are cheap to synthesize into proteins that are highly expressed. Such selection pressure has been demonstrated to alter amino acid usage in a few organisms, but whether “cost selection” is a general phenomenon remains unknown. One reason for this is that reliable protein expression level data is not available for most organisms. Accordingly, I have developed a new method for detecting cost selection. This method depends solely on interprotein gradients in amino acid usage. Applying it to an analysis of 43 whole genomes from all three domains of life, I show that selection on the synthesis cost of amino acids is a pervasive force in shaping the composition of proteins. Moreover, some amino acids have different price tags for different organisms—the cost of amino acids is changed for organisms living in hydrothermal vents compared with those living at the sea surface or for organisms that have difficulty acquiring elements such as nitrogen compared with those that do not—so I also investigated whether differences between organisms in amino acid usage might reflect differences in synthesis or acquisition costs. The results suggest that organisms evolve to alter amino acid usage in response to environmental conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Hector Musto]     

Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol

Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H⁺ conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l^?1 of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.     

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.     

In silico deletion of PtsG gene in Escherichia coli genome-scale model predicts increased succinate production from glycerol

Systems metabolic engineering and in silico analyses are necessary to study gene knockout candidate for enhanced succinic acid production by Escherichia coli. Metabolically engineered E. coli has been reported to produce succinate from glucose and glycerol. However, investigation on in silico deletion of ptsG/b1101 gene in E. coli from glycerol using minimization of metabolic adjustment algorithm with the OptFlux software platform has not yet been elucidated. Herein we report what is to our knowledge the first direct predicted increase in succinate production following in silico deletion of the ptsG gene in E. coli GEM from glycerol with the OptFlux software platform. The result indicates that the deletion of this gene in E. coli GEM predicts increased succinate production that is 20% higher than the wild-type control model. Hence, the mutant model maintained a growth rate that is 77% of the wild-type parent model. It was established that knocking out of the ptsG/b1101 gene in E. coli using glucose as substrate enhanced succinate production, but the exact mechanism of this effect is still obscure. This study informs other studies that the deletion of ptsG/b1101 gene in E. coli GEM predicted increased succinate production, enabling a model-driven experimental inquiry and/or novel biological discovery on the underground metabolic role of this gene in E. coli central metabolism in relation to increasing succinate production when glycerol is the substrate.     

Intracytoplasmic membrane production inEscherichia coli O111a1: Nutritional parameters

Escherichia coli O111a₁ ceased growth prematurely and accumulated intracytoplasmic membrane at 42°C in an amino acids-mineral salts medium. The amount of membrane formed appeared to be proportional to the concentration of amino acids in the medium—the greater the concentration of amino acids in the medium, the greater the membrane production.E. coli O111a₁, did not grow at 42°C in glucose-, glycerol- or acetate-mineral salts medium, but mesosome-like structures were produced in glucose-grown cells and some intracytoplasmic membrane in cells grown on glycerol and acetate. Supplementation of the glucose medium with pantothenate and/or thiamine permitted normal growth. The vitamins did not restore growth of the mutant in glycerol or acetate, but intracytoplasmic membrane production was increased, especially in glycerol. Amino acids plus glucose supported normal growth with no membrane production. Glycerol and acetate had no effect on the growth in the amino acids medium, but stimulated the accumulation of membrane.     

Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H⁺ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l^?1 (41.2-fold in 7 days) from glycerol and .39 g l^?1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.     

Amino acid transport and protein synthesis in energetically-stable calcium-tolerant isolated cardiac myocytes

Myocytes isolated by enzymic dispersion from adult rat ventricular tissue are shown to be energetically stable in the presence of 0.5 mM CaCl₂. ATP and ADP content and rates of lactate production are comparable with those of intact myocardial tissue and consistent with these cells being tightly coupled. Addition of 2,4-dinitrophenol precipitates rapid changes in adenine nucleotide concentrations and a 10-fold increase in lactate production. Cardiac myocytes selectively transport neutral amino acids of the A and L classes. Transport of the amino acid analogue α-aminoisobutyric acid is an active, temperature-dependent and insulin-sensitive process. The apparent K_m for α-aminoisobutyric acid transport is similar to that determined for embryonic cardiac cells. Mature myocytes incorporate labelled amino acids into cytoplasmic proteins with molecular weights ranging from 10 000 to 150 000. Newly synthesised protein is metabolically stable. The data establishes calcium-tolerant myocytes as an experimental system offering many advantages over whole hearts for short- and long-term studies of protein synthesis and catabolism.     

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism

In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L^?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L^?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L^?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L^?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L^?1 during test tube cultivation.

     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。     

Evolutionarily Conserved Optimization of Amino Acid Biosynthesis

The “cognate bias hypothesis” states that early in evolutionary history the biosynthetic enzymes for amino acid x gradually lost residues of x, thereby reducing the threshold for deleterious effects of x scarcity. The resulting reduction in cognate amino acid composition of the enzymes comprising a particular amino acid biosynthetic pathway is predicted to confer a selective growth advantage on cells. Bioinformatic evidence from protein-sequence data of two bacterial species previously demonstrated reduced cognate bias in amino acid biosynthetic pathways. Here we show that cognate bias in amino acid biosynthesis is present in the other domains of life—Archaebacteria and Eukaryota. We also observe evolutionarily conserved underrepresentations (e.g., glycine in methionine biosynthesis) and overrepresentations (e.g., tryptophan in asparagine biosynthesis) of amino acids in noncognate biosynthetic pathways, which can be explained by secondary amino acid metabolism. Additionally, we experimentally validate the cognate bias hypothesis using the yeast Saccharomyces cerevisiae. Specifically, we show that the degree to which growth declines following amino acid deprivation is negatively correlated with the degree to which an amino acid is underrepresented in the enzymes that comprise its cognate biosynthetic pathway. Moreover, we demonstrate that cognate fold representation is more predictive of growth advantage than a host of other potential growth-limiting factors, including an amino acid’s metabolic cost or its intracellular concentration and compartmental distribution. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Niles Lehman Ethan O. Perlstein and Benjamin L. de Bivort contributed equally to this work.     

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Benjamin L. de Bivort and Ethan O. Perlstein have contributed equally to this work.     

A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization,Gene Cloning and Primary Structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.