Metabolic engineering of Saccharomyces cerevisiae for conversion of D-glucose to xylitol and other five-carbon sugars and sugar alcohols

Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar D-ribose from D-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated D-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (D-ribose, D-ribulose, and D-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted.     

Metabolic engineering of sugar catabolism in lactic acid bacteria

Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.     

Metabolic engineering

Metabolic engineering has developed as a very powerful approach to optimising industrial fermentation processes through the introduction of directed genetic changes using recombinant DNA technology. Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes. In this paper, some of these tools are reviewed and many examples of metabolic engineering are presented to illustrate the power of the technology. The examples are categorised according to the approach taken or the aim: (1) heterologous protein production, (2) extension of substrate range, (3) pathways leading to new products, (4) pathways for degradation of xenobiotics, (5) improvement of overall cellular physiology, (6) elimination or reduction of by-product formation, and (7) improvement of yield or productivity.     

Accumulation of sugar alcohols by filamentous fungi

Five hundred isolates of different xerophilic and non-xerophilic fungi belonging to 10 genera and 74 species were screened for alditol (sugar alcohol) accumulation. Ninety-two of the isolates failed to grow on a salt medium, most of the isolates (408) produced alditols; 348,44 and 16 of them produced low, moderate and high levels of alditols, respectively. The high alditol producers belonged to five species ofAspergillus, six species ofEurotium andFennellia flavipes. Glycerol andd-mannitol were the main constituents of alditol pools of the 16 high alditol producers.d-Arabinitol andmeso-erythritol were also formed but at low concentrations by several of the tested isolates.     

Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.     

Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Metabolic engineering of bacteria

Yield and productivity are critical for the economics and viability of a bioprocess. In metabolic engineering the main objective is the increase of a target metabolite production through genetic engineering. Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the production of a certain substance. In the last years, the development of recombinant DNA technology and other related technologies has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathway. Industrial microorganisms like Escherichia coli, Actinomycetes, etc. have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. The factors like oxygenation, temperature and pH have been traditionally controlled and optimized in industrial fermentation in order to enhance metabolite production. Metabolic engineering of bacteria shows a great scope in industrial application as well as such technique may also have good potential to solve certain metabolic disease and environmental problems in near future.     

Metabolic engineering for improved microbial pentose fermentation

Global concern over the depletion of fossil fuel reserves, and the detrimental impact that combustion of these materials has on the environment, is focusing attention on initiatives to create sustainable approaches for the production and use of biofuels from various biomass substrates. The development of a low-cost, safe and eco-friendly process for the utilization of renewable resources to generate value-added products with biotechnological potential as well as robust microorganisms capable of efficient fermentation of all types of sugars are essential to underpin the economic production of biofuels from biomass feedstocks. Saccharomyces cerevisiae, the most established fermentation yeast used in large scale bioconversion strategies, does not however metabolise the pentose sugars, xylose and arabinose and bioengineering is required for introduction of efficient pentose metabolic pathways and pentose sugar transport proteins for bioconversion of these substrates. Our approach provided a basis for future experiments that may ultimately lead to the development of industrial S. cerevisiae strains engineered to express pentose metabolising proteins from thermophilic fungi living on decaying plant material and here we expand our original article and discuss the strategies implemented to improve pentose fermentation.     

Metabolic engineering in silico

This review briefs on the main directions in the field of mathematical modeling of metabolic processes aimed at a rational design of genetically modified organisms. The class of generalized Hill functions is described, and their application to modeling of nonlinear processes in Escherichia coli metabolic systems is illustrated by several examples. A model for the pyrimidine biosynthesis in E. coli, taking into account the nonlinear effects of a negative allosteric regulation of enzyme activities involved in the control of the subsequent stages by the end products of synthesis, is considered. It has been shown that the model displays its own continuous oscillation mode of functioning with a period of approximately 50 min, which is close to the duration of E. coli cell cycle. The need in considering the nonlinear effects in the models as essential elements in the function of metabolic systems far from equilibrium is discussed.     

Metabolic engineering of Propionibacterium freudenreichii for n-propanol production

Propionibacteria are widely used in industry for manufacturing of Swiss cheese, vitamin B₁₂, and propionic acid. However, little is known about their genetics and only a few reports are available on the metabolic engineering of propionibacteria aiming at enhancing fermentative production of vitamin B₁₂ and propionic acid. n-Propanol is a common solvent, an intermediate in many industrial applications, and a promising biofuel. To date, no wild-type microorganism is known to produce n-propanol in sufficient quantities for industrial application purposes. In this study, a bifunctional aldehyde/alcohol dehydrogenase (adhE) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. The mutants expressing the adhE gene converted propionyl- coenzyme A, which is the precursor for propionic acid biosynthesis, to n-propanol. The production of n-propanol was limited by NADH availability, which was improved significantly by using glycerol as the carbon source. Interestingly, the improved propanol production was accompanied by a significant increase in propionic acid productivity, indicating a positive effect of n-propanol biosynthesis on propionic acid fermentative production. To our best knowledge, this is the first report on producing n-propanol by metabolically engineered propionibacteria, which offers a novel route to produce n-propanol from renewable feedstock, and possibly a new way to boost propionic acid fermentation.     

Metabolic engineering of Candida utilis for isopropanol production

A genetically-engineered strain of the yeast Candida utilis harboring genes encoding (1) an acetoacetyl-CoA transferase from Clostridium acetobutylicum ATCC 824, (2) an acetoacetate decarboxylase, and (3) a primary–secondary alcohol dehydrogenase derived from Clostridium beijerinckii NRRL B593 produced up to 0.21 g/L of isopropanol. Because the engineered strain accumulated acetate, isopropanol titer was improved to 1.2 g/L under neutralized fermentation conditions. Optimization of isopropanol production was attempted by the overexpression and disruption of several endogenous genes. Simultaneous overexpression of two genes encoding acetyl-CoA synthetase and acetyl-CoA acetyltransferase increased isopropanol titer to 9.5 g/L. Moreover, in fed-batch cultivation, the resultant recombinant strain produced 27.2 g/L of isopropanol from glucose with a yield of 41.5 % (mol/mol). This is the first demonstration of the production of isopropanol by genetically engineered yeast.     

Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation

Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.     

Metabolic engineering for microbial production of shikimic acid

Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections. An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms. Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid. An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate. Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.     

Metabolic engineering of Corynebacterium glutamicum for L-serine production

Although L-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via L-serine formation, previous attempts to obtain a strain producing L-serine from glucose have not been successful. We functionally identified the genes serC and serB from Corynebacterium glutamicum, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic serA gene, serA(delta197), encoding an L-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of L-serine, as did the overexpression of these genes in a strain with the L-serine dehydratase gene sdaA deleted. However, reduced expression of the serine hydroxymethyltransferase gene glyA, in combination with the overexpression of serA(delta197), serC, and serB, resulted in a transient accumulation of up to 16 mM L-serine in the culture medium. When sdaA was also deleted, the resulting strain, C. glutamicum delta sdaA::pK18mobglyA'(pEC-T18mob2serA(delta197)CB), accumulated up to 86 mM L-serine with a maximal specific productivity of 1.2 mmol h(-1) g (dry weight)(-1). This illustrates a high rate of L-serine formation and also utilization in the C. glutamicum wild type. Therefore, metabolic engineering of L-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism.     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.