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PubMed2002

Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering.

Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements. As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of Clostridium cellulolyticum, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight. The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively. This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered C. cellulolyticum.

Alcohol Dehydrogenase↗
PubMed2002

Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.

Acetaldehyde↗
PubMed2002

Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis.

Morphology and alpha-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of Aspergillus oryzae in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The alpha-amylase productivity was not significantly different in the three strains. A strain in which the transcription of chsB could be controlled by the nitrogen source-regulated promoter niiA (NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for chsB in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect alpha-amylase productivity. alpha-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed.

Aspergillus oryzae↗
PubMed2002

Metabolic engineering of glycerol production in Saccharomyces cerevisiae.

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.

Cell Culture Techniques↗
PubMed2003

Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110.

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of L-lactic acid to D-lactic acid. Although the largest demand is for the L enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of D-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. D-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this D-lactic acid was approximately 98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of D-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.

Acetates↗
PubMed2003

Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis.

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

Animals↗
PubMed2003

Increased production of folate by metabolic engineering of Lactococcus lactis.

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.

Bacterial Proteins↗
PubMed2003

High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis.

Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 micro mol/liter, which is close to industrial vitamin B(12) production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of E. coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.

5-Aminolevulinate Synthetase↗
PubMed2004

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii.

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.

Arabidopsis↗
PubMed2004

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae.

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.

Aldehyde Oxidoreductases↗
PubMed2005

High-level acetaldehyde production in Lactococcus lactis by metabolic engineering.

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.

Acetaldehyde↗
PubMed2006

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway.

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.

Bacterial Proteins↗
PubMed2006

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a hexachlorobenzene degrader by metabolic engineering.

The gene cassette (camA+ camB+ camC) encoding a cytochrome P-450cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol.mg (dry weight)-1.h-1, and intermediate pentachlorophenol was also identified.

Bacterial Proteins↗
PubMed2006

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase.

BACKGROUND: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. RESULTS: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. CONCLUSION: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

Carotenoids↗
PubMed2002

Bacterial autoinduction: looking outside the cell for new metabolic engineering targets.

Recent evidence has demonstrated that cell-to-cell signaling is a fundamental activity carried out by numerous microorganisms. A number of specialized processes are reported to be regulated by density-dependent signaling molecules including antibiotic production, bioluminescence, biofilm formation, genetic competence, sporulation, swarming motility and virulence. However, a more centralized role for quorum sensing is emerging where quorum signaling pathways overlap with stress and starvation circuits to regulate cellular adaptation to changing environmental conditions. The interplay of these phenomena is especially critical in the expression of recombinant proteins where elicitation of stress responses can dramatically impact cellular productivity.

Journal Article↗
PubMed2006

Metabolic engineering of Saccharomyces cerevisiae producing nicotianamine: potential for industrial biosynthesis of a novel antihypertensive substrate.

Nicotianamine (NA), a metal chelator, is ubiquitous in higher plants. In humans, NA inhibits angiotensin I-converting enzyme (ACE), and consequently reduces high blood pressure. Nicotianamine is synthesized from the trimerization of S-adenosylmethionine (SAM) by NA synthase (NAS). Here, we aimed to produce large amounts of NA fermentatively by introducing the Arabidopsis AtNAS2 gene into Saccharomyces cerevisiae strain SCY4. This strain can accumulate up to 100 times the usual amount of SAM, and this is considered desirable for overproduction of NA. Nicotianamine was produced in the engineered yeast, and the NA level increased with incubation time until the stationary phase. The maximum concentration of intracellular NA obtained was 766+/-33 microg/g wet weight. Successful production of NA in S. cerevisiae should pave the way for industrial production of this novel antihypertensive substrate.

Alkyl and Aryl Transferases↗
PubMed1994

Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae.

We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of beta-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. beta-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.

Carotenoids↗