Conversion of proteins into biofuels by engineering nitrogen flux

Biofuels are currently produced from carbohydrates and lipids in feedstock. Proteins, in contrast, have not been used to synthesize fuels because of the difficulties of deaminating protein hydrolysates. Here we apply metabolic engineering to generate Escherichia coli that can deaminate protein hydrolysates, enabling the cells to convert proteins to C4 and C5 alcohols at 56% of the theoretical yield. We accomplish this by introducing three exogenous transamination and deamination cycles, which provide an irreversible metabolic force that drives deamination reactions to completion. We show that Saccharomyces cerevisiae, E. coli, Bacillus subtilis and microalgae can be used as protein sources, producing up to 4,035 mg/l of alcohols from biomass containing ～22 g/l of amino acids. These results show the feasibility of using proteins for biorefineries, for which high-protein microalgae could be used as a feedstock with a possibility of maximizing algal growth and total CO(2) fixation.     

Kinetics of amino acid incorporation into serum proteins

1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q₁₀ was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q₁₀ of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

Incorporation of cystine into the proteins of regenerating wound tissue

A procedure to distinguish between cystine bound into protein by peptide bonds and that associated with the protein as prosthetic groups by disulfide bonds only has been devised. This distinction is achieved by reaction of the cysteine and cystine in the protein with sodium sulfite in alkaline medium and simultaneous separation of the resulting thiosulfate derivatives by dialysis. Of the total cystine in regenerating wound tissue proteins, 6.6% is bound by disulfide bonds only. This fraction of the half-cystine residues turns over very much more rapidly than do the cystine residues in the protein bound by peptide bonds. The formation and turnover of liver proteins, as indicated by cystine-S³⁵ incorporation, is much more rapid in wounded than in normal rats. Wounding also appears to have the effect of increasing the proportion of half-cystine residues linked to the liver proteins by disulfide bonds only.     

Comparative evaluation of the action of serum proteins on tissue regeneration

Processes of skin and long tubular bones' reparative regeneration under the action of serum albumin and alpha-fetoprotein on the body were studied with the use of EPR and histological methods. Relations of spin probe rotatory mobility in cell suspensions and morphological change of examined tissues are shown. It is established that the biological activity of alpha-fetoprotein is more pronounced than that of serum albumin in the effect on the young connective tissue. The stimulating effect of the embryonic protein is demonstrated only in the first week of reparative process while that of serum albumin appeared much more late.     

Hyperglycemia and non-enzymatic glycation of serum and tissue proteins in chickens

The objectives of these studies were to determine whether elevated plasma glucose concentrations in broiler breeder chickens (200–250 mg/dl) can result in the non-enzymatic attachment of glucose to serum proteins (fructosamine) and eventual cross-linking of tissue proteins (basement membrane thickness), and to investigate the effects of a factor that may influence this cross-linking process. In response to feeding the satiety factor calcium propionate (CaP, 1.7%), plasma glucose and fructosamine concentrations were increased (P < 0.05) from 1 to 9 weeks of age, whereas concentrations of plasma glucose and fructosamine in feed-restricted chicks were reduced for the first 7 weeks after hatch. In a second study, the age-related increase in kidney capillary basement membrane thickness was prevented (P < 0.05) by feeding the cross-linking inhibitor aminoguanidine (AG, 800 ppm) to 30-week-old broiler breeder hens for 34 weeks.The results from these studies suggest that concentrations of plasma glucose in chickens may, in fact, be exerting long-term detrimental effects on tissue proteins, which can be ameliorated by factors that limit the cross-linking reaction.     

Leucine catabolism and incorporation into tissue proteins in thyroparathyroidectomized rats

We investigated parameters of leucine metabolism in thyroparathyroidectomized (TPX) and pair-fed control rats using a technique of continuous infusion of [l-14C]leucine. The rate of leucine turnover was significantly smaller in TPX than in control rats (42.5 +/- 2.6 vs 35.1 +/- 1.9 mumole/hr/100 g, mean +/- SEM, six rats). There was no significant difference between rates of alpha-decarboxylation of leucine by the two groups of rats. The protein incorporation of leucine was significantly smaller in the muscle of TPX than control rats (39 +/- 5 vs 24 +/- 4 pmole/mg protein, mean +/- SEM, six rats) but in liver it was not significantly different. Thyroparathyroidectomy also had no significant effect on concentration of either leucine or its ketoacid (alpha-ketoisocaproate) in plasma, liver, and muscle. We conclude that hypothyroidism does not alter catabolism of leucine but reduces its incorporation into muscle protein.     

Proteinuria in neonatal piglets after oral and intravenous administration of homologous colostrum and serum proteins

• 1.1. Proteinuria was induced in piglets (0–45 days after birth) by administration of sow colostrum and porcine serum intravenously or orally.
• 2.2. Electroimmunoassay and Sephadex gel nitration was used to quantitate and follow the fate in the organism of total protein, albumin, α-foetoprotein and colostrum prealbumin and to determine the molecular weights of these specific proteins.
• 3.3. Light microscopy and electronmicroscopy were used to study changes in vesicular activity in the proximal tubular cells during naturally occurring and induced proteinuria.
• 4.4. The transient proteinuria developed by the newborn piglet after ingestion of colostrum seems to be an effect of changes in amount and quality of protein on the capillary side of the glomerular cells and in function of the proximal tubular cells of the kidney.

     

Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells

Microinjection of physiologic quantities of macromolecules into tissue culture cells can facilitate the study of the biological effects of such macromolecules. In this communication, we describe a chemical technique which can be used to microinject proteins into monolayers of intact cells. Protein is loaded into erthrocyte ghosts, and the ghosts are then fused to the monolayer with polyethylene glycol 1000. Receipient cells can be injected with an efficiency of greater than 90% and contain an average of 3.8 X 10(6) microinjected molecules per cell. This technique circumvents certain problems encountered in virus-induced microinjection.     

Conversion of Cellobiose into Lactose

Benzyl 2, 3, 6-tri-O-acetyl-4-O-(2,3-di-O-acetyl-4,6-di-O-methylsulfonyl-β-d-glucopyranosyl)-β-d-glucopyranoside (VI) was prepared from α-cellobiose octaacetate. Displacement of the sulfonyl esters of VI with acyloxy-groups in N, N-dimethyl formamide in the presence of sodium benzoate gave 4-O-β-d-galactopyranosyl-d-glucopyranose derivative (lactose derivative). Elimination of blocking groups of the derivative yielded lactose hydrate (IX), though the overall yield of lactose from cellobiose octaacetate was less than 2%.     

Conversion of 14C-galactose into amino acids in tissue culture cells and its inhibition by manganese

The incorporation of 14C-galactose into primary AGMK-cells was studied in the presence and absence of Mn2+. The transport of galactose into the cells is not influenced by Mn2+. 1 mM MnCl2 inhibits the incorporation of galactose into acid-precipitable material up to 50% after 6 hours incubation. In the absence of Mn2+ a substantial amount of galactose is converted to glucose, which is mainly metabolized into aspartic acid and serine. The conversion of galactose into glucose is inhibited by the addition of Mn2+. However, Mn2+ does not influence the activity of the UDP-galactose-4'-epimerase in vitro. Using the SDS-polyacrylamide electrophoresis the labelling of protein bands is similar with 14C-galactose or a 14C-amino acid mixture, respectively. In the presence of Mn2+ the incorporation of both galactose or amino acids is inhibited: With amino acids the inhibition is observed in all protein bands, whereas with galactose some bands remain unaffected. It is concluded that these are galactoproteins.     

Conversion of Coccidioides immitis in tissue culture

Summary Utilization of a pre-treatment resulted in the establishment of a simple technique to enhance spherulation ofC. immitis in tissue culture.Establishment of the fact that culture spherules are produced from mycelial elements was also recognized and should be included in the life cycle ofC. immitis when cultured in vitro.