Bulk chemical production by metabolically engineered
Abstract
Sustainable production of chemicals is of increasing importance, due to depletion of petroleum and environmental concerns. In addition to its importance in basic research as a simple, eukaryotic model organism, Saccharomyces cerevisiae has long been exploited in industry because of its physiological properties. And today, the development in genetic engineering toolbox and genome-scale metabolic models of S. cerevisiae has extended its application range to new products and bioprocesses. In addition, evolutionary engineering strategies have been useful in improving cellular properties of S. cerevisiae, such as tolerance to product toxicity and inhibitors. In this chapter, recent metabolic and evolutionary engineering studies that involve S. cerevisiae for the production of bulk chemicals and fine chemicals including flavours and pharmaceuticals are reviewed. It was shown that metabolic engineering particularly allowed the improvement of pharmaceuticals production, which will enable economic and large-scale production of many valuable pharmaceuticals. It is clear that S. cerevisiae will continue to be an important host for future metabolic engineering and metabolic pathway engineering applications to produce a variety of industrially and clinically important chemicals.
Keywords
- pharmaceuticals
- adaptive evolution
- bulk chemicals
- evolutionary engineering
- flavours
- fine chemicals
- glutathione
- metabolic engineering
- organic acids
- Saccharomyces cerevisiae
1. Introduction
Metabolic engineering was defined by Bailey [1] as ‘the improvement of cellular activities by manipulation of enzymatic, transport and regulatory functions of the cell with the use of recombinant DNA technology’. More than 20 years after this first definition as a new scientific discipline, metabolic engineering has become an increasingly important research field of biotechnology. Today, metabolic engineering requires interdisciplinary work that includes molecular biology, applied microbiology, biochemical reaction engineering, biomedical research with the aid of high-throughput analytical tools in ‘omics’ research and bioinformatics [2].
There are two major approaches in metabolic engineering, as described by Bailey et al. [3], the rational metabolic engineering and inverse metabolic engineering. In rational metabolic engineering, extensive genetic and biochemical information is required on the metabolism or metabolic pathway of interest to make defined genetic manipulations. The cellular physiological responses are also complex. Thus, trying to re-engineer a cellular machine that is too complex and about which there is limited information is a major limitation in rational metabolic engineering. Difficulties in cloning in industrial strains due to the lack of relevant genetic tools, and GMO-concerns of the public regarding food industry are additional issues [2]. The inverse metabolic engineering approach was designed to avoid the above-mentioned limitations. Here, the desired phenotype is identified first, as a ‘bottom-up’ approach, and then its genetic and/or environmental basis is determined which is the most challenging step. However, owing to the powerful high-throughput analytical technologies in genomics, transcriptomics, proteomics and metabolomics, this step is becoming easier [2, 4]. Thus, without any need for extensive initial information on biochemistry, genetics and regulation on the organism of interest, the desired phenotype can be obtained. Adaptive evolution or evolutionary engineering, which is based on random mutation and selection by systematic cultivation of an initial microbial culture in the presence of a selective pressure to obtain desirable phenotypes [5], is a common inverse metabolic engineering strategy [2].
Metabolic engineering is a key strategy for harnessing microorganisms’ ability to produce chemicals from renewable carbon sources. Microbial processes are attractive since they have significantly lower environmental impacts than the petroleum-based processes. However, the former is primarily an economic challenge. Therefore, it is vital to develop superior strains with improved yield, titer and productivity by engineering microbial physiology, stress response and metabolism [6]. Considering the market value of chemical products based on petroleum, the cost-competitive bio-based products, once achieved, would have significant economic value as replacements. It is estimated that the global market share of bio-based chemicals will rise from 2% in 2008 to 22% in 2025 [7].
In this chapter, we focused on the recent metabolic engineering studies that involve the baker’s yeast,
2. Production of bulk chemicals
The oil refinery is currently the major source of bulk chemicals such as solvents and polymer precursors. A significant portion of petroleum is used in the chemical catalysis for the production of chemicals and plastics [8]. However, in recent years, microbial production of chemicals based on renewable sources, such as biomass, has become important as a part of the efforts to reduce demand on diminishing petroleum and to reduce hazardous wastes. In addition, biotechnology makes new chemical monomers accessible, which are otherwise inaccessible due to high production cost [9].
In bio-refineries, the biomass is the first converted into simple sugars and then to valuable chemicals. Microorganisms are the main players of the latter conversion. Therefore, the development of a suitable strain for the particular process is needed. As a model yeast,
Bulk chemical produced | Representative studies and their strain improvement strategy [reference no] |
---|---|
Succinic acid | Disturbance of the citric acid cycle by deleting Disabled serine synthesis from glycolysis through a triple deletion of Enhanced succinic acid export via heterologous expression of |
Itaconic acid | Overexpression of |
3-Hydroxypropionic acid | Reconstruction of malonyl-CoA to 3-HP pathway via expression of Reconstruction of β-alanine to 3-HP pathway via coexpression of Reconstruction of malonyl-CoA to 3-HP pathway via coexpression of Adaptive laboratory evolution for improved tolerance to 3-HP at pH 3.5 [45] |
Lactic acid | Expression of genome-integrated Expression of genome-integrated Deletion of Inhibition of L-LDH consumption by deletion of Overexpression of Repression of ethanol production by deleting Enhancement of lactic acid transport by expressing Expression of |
Succinic acid is used in a wide range of industries from food to agriculture. Also, it has been considered as a generic intermediate for the bio-based polymers and can be a substitute of petroleum-derived maleic anhydride, which has a huge market [11]. Therefore, an increasing demand of succinic acid is expected in the future. Currently, it is mainly produced by chemical syntheses, which are based on petrochemical precursors. Biotechnological routes are pursued to achieve a sustainable production of succinic acid.
Itaconic acid has currently application in the manufacture of pharmaceuticals, adhesives and resins. In addition, its polymerized form (polyitaconic acid) has potentials as a replacement of acrylic acid in the development of superabsorbents [17], and can be used in contact lenses, detergents and cleaners [18].
3-Hydroxypropionic acid (3-HP) is another important platform chemical which can be produced from either sugars or glycerol and can be converted to 1,3-propanediol, acrylic acid, malonic acid, and acrylamide. 3-HP derivatives have a variety of applications in super absorbent polymers, surface coatings, adhesives and paints [11]. Although there are biological pathways to 3-HP via either glycerol, lactate, malonyl-CoA or β-alanine intermediates, no organism is known to produce it as an end product [22]. The pathways based on malonyl-CoA and β-alanine have been constructed in
Lactic acid is a well-known fermentation product which is already widely used in food, cosmetics and pharmaceutical industries. Lactic acid derived from biomass is also valued as a monomer in the development of bioplastics [26]. Lactic acid bacteria, especially,
Product toxicity is a major obstacle for achieving high titers of the target chemicals such as organic acids, aromatic substances and antibiotics [36]. There is limited knowledge about the molecular basis of the product toxicity and tolerance to enable a rational prediction of genetic changes [37]. David et al. developed, for the first time, a hierarchical dynamic pathway control system involving a two-stage fermentation concept and the use of a metabolic sensor in
3. Production of fine chemicals
Plant secondary metabolites hold the potential to be used as pharmaceuticals, cosmetic and food ingredients. However, the yield of these molecules when extracted from natural producers is not in sufficient amounts to meet industrial demands. In addition, chemical synthesis of these complex structures often requires multiple reaction steps and is not a commercially attractive route due to low product yields [46]. Currently, advances in metabolic engineering allowed commercial-scale microbial production of a number of fine chemicals [47–49]. Besides, there is an ongoing academic interest for reconstitution of biosynthetic pathways of several natural products, including complex pathways, in
3.1. Flavours
Compounds belonging to isoprenoid and phenolics type of secondary metabolites are valued as natural fragrances and flavours. Flavour compounds can be produced from sugars (
Vanillin, a phenolic aldehyde, is one of the first flavour compounds produced in microbial hosts at commercial-scale. Current state of the microbial production of vanillin based on various precursors and the available production hosts have been recently reviewed by Gallage and Møller [52].
β-Ionone is an apocarotenoid that is naturally present in raspberries. In
2-Phenyl ethanol (2-PE) is another economically attractive flavour compound with a rose-like scent. Ehrlich pathway is involved in the bioconversion of phenylalanine to 2-phenyl ethanol within
3.2. Pharmaceuticals
Another major area of metabolic engineering research is the production of clinically important compounds. In this section, examples will be given for the production of a variety of such compounds by metabolically engineered yeast. Representative examples for the production of pharmaceuticals by metabolically engineered
Pharmaceutical produced | Representative studies and their strain improvement strategy [reference no] |
---|---|
Glutathione | Overexpression of Manipulation of the sulphate assimilation pathway by overexpressing Improved oxidized glutathione production by overexpression of Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuffling and screening for enhanced glutathione production [62] |
Artemisinin/artemisinic acid | Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of |
Taxol/taxadiene | Expression of a truncated version of the endogenous Prediction of the efficiency of different GGPPS enzymes via computer aided protein modelling [67] |
Forskolin | Expression of a promiscuous cytochrome P450 from |
Polyketides | Heterologous expression of 6-MSA synthase gene from Construction of polyketide precursor pathways by expressing Enhanced cofactor supply by expressing |
Resveratrol | Reconstruction of a Expression of Expression of Expression of 4-coumaroyl-coenzyme A ligase ( Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] |
Dihydrochalcones | Expression of the heterologous pathway genes in a |
Alkaloids | Expression of 14 monoterpene indole alkaloid pathway genes from Construction of the complete Expression of AdoMet-dependent methyltransferase enzymes (6-OMT, CNMT and 4’-OMT) from plant and human origin to produce reticuline from norlaudanosoline [81] Reconstruction of berberine biosynthetic pathway from reticuline by expressing seven relevant heterologous genes [82] Reconstruction of a 10-gene biosynthetic pathway from plant to produce sanguinarine from norlaudanosoline [83] Expression of 16 heterologous plant enzymes to produce noscapine from canadine [84] Reconstruction of a seven-gene pathway for the production of codeine and morphine from ( Reconstruction of a |
Glutathione, a naturally occurring tripeptide, is an important compound used in health and cosmetic industries. It is produced by using
Terpene derivatives are economically viable molecules that are used in the synthesis of drugs such as the antimalarial agent artemisinin, and the anticancer agent taxol [63]. Several terpenoids have been produced in
The well-known diterpenoid taxol is an anti-cancer agent [63, 65]. As a first step towards taxol production,
Forskolin is a labdene diterpene with potentials to be used in the treatment of blood pressure, in weight-loss supplements and in the protection against congestive heart failure. Ignea et al. constructed a yeast platform to produce 11β-hydroxy-manoyl oxide, forskolin precursor. Although the forskolin biosynthetic pathway has not been completely discovered yet, a promiscuous cytochrome P450 from
Polyketides are also a major group of natural products with a wide range of applications as antibiotics, immunosuppressors, cholesterol lowering agents and other drugs [69].
The strategy of engineered precursor pools has also been applied in the production of resveratrol. Resveratrol is a polyketide derivative with potent antioxidant properties and it has been recently brought to market as a bio-product [72]. Earlier reports on the production of resveratrol were based on bioconversion of aromatic precursors such as
Dihydrochalcones (DHCs) such as nothofagin, phlorizin and naringin dihydrochalcone are another group of polyketide derivatives with commercial value as antioxidants, antidiabetics or sweeteners. Recently,
Recently, there have also been many reports on the reconstitution of biosynthetic pathways of alkaloids in yeast. Alkaloids are nitrogen-containing complex molecules with potent biological activity. Currently, there are around 50 alkaloid-based drugs, including the anticancer drug vincristine, the antitussive agent noscapine and the analgesic codeine. Strictosidine was the first reported plant-derived alkaloid produced
4. Summary and outlook
For fine chemicals such as amino acids, vitamins, flavours, nutraceuticals, organic acids and fragrances, profit margins are usually not high and could be affected by substrate availability and cost. However, metabolic engineering enabled improvements in production of both pharmaceuticals and fine chemicals which will allow economic and large-scale production of many valuable compounds in near future.
It is obvious that
Acknowledgments
We thank Arman Akşit for technical assistance. Our research presented in this review was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (project nos: 105T314 and 107T284, PI: ZPÇ), TUBITAK-COST (project no: 109T638, PI: ZPÇ, COST Action no: CM0902), Federation of European Microbiological Societies (FEMS) Research Fellowship (2009-2 to BTY), Turkish State Planning Organization (DPT) and Istanbul Technical University Research Funds (project nos: 30108, 33237, 34200; PI: ZPÇ). BH is financially supported by the Faculty Member Training Programme (ÖYP) of the Council of Higher Education (YÖK), Turkey. We also would like to thank former graduate students Urartu Ö.Ş. Şeker, Ceren Alkım, Tuğba Sezgin, Ülkü Yılmaz, Gökhan Küçükgöze and Berrak Gülçin Balaban, as well as our colleagues and collaborators Uwe Sauer, Jean Marie François, Laurent Benbadis, Süleyman Akman, Bülent Balta, Candan Tamerler and Mehmet Sarıkaya for their scientific contribution in our research presented in this review.
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