Advances in metabolic engineering of yeast<i>Saccharomyces cerevisiae</i>for production of chemicals

Borodina, Irina; Nielsen, Jens

doi:10.1002/biot.201300445

Cited by 239 publications

(167 citation statements)

References 74 publications

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“…Yeasts, and in particular S. cerevisiae, are widely used as a cell factory for production of various chemicals (83). However, all known TAL enzymes of fungal origin also have at least a partial PAL activity.…”

Section: Resultsmentioning

confidence: 99%

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Jendresen

Stahlhut

et al. 2015

Appl Environ Microbiol

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Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 M cinnamic acid per unit of optical density at 600 nm [OD 600 ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 M p-coumaric acid OD 600 unit ؊1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. Small organic molecules of biotechnological interest include aromatic structures that are derived from p-coumaric acid (pHCA). pHCA can be formed from phenylalanine either through deamination to cinnamic acid (CA) by phenylalanine ammonialyase (PAL; EC 4.3.1.24) and subsequent hydroxylase activity by trans-cinnamate-4-monooxygenase or through deamination of tyrosine by tyrosine ammonia-lyase (TAL; EC 4.3.1.23) (Fig. 1). Considering that the route from phenylalanine requires activity of a P450 enzyme, which has been shown to be the limiting step in previous engineering strategies (1, 2), deamination of tyrosine for production of pHCA may be preferred in microbial cell factories. Tyrosine ammonia-lyases (TAL) have been employed for the production of plant biochemicals and aromatic compounds, e.g., for pHCA itself (3-5), or in the production of stilbenes, such as resveratrol (6, 7), flavonoids, such as naringenin (8, 9), cinnamoyl anthranilates (10), or plastic precursors, such as p-hydroxystyrene (11, 12), yet the TAL reaction may be the limiting step (10, 13).Due to the significant interest in production of phenolic compounds in biotechnological processes, we aimed at increasing the range of characterized phenylalanine ammonia-lyases (PAL) and tyrosine ammonia-lyases (TAL), and in particular at identifying the optimal TAL for use in microbial cell factories...

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Section: Resultsmentioning

confidence: 99%

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Jendresen

Stahlhut

et al. 2015

Appl Environ Microbiol

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“…The strategies for metabolic engineering to increase productivity have been commonly focused on improvement of the downstream synthesis pathway of the target chemicals as well as repression of the competitive pathways that form by-products (16)(17)(18). In addition, enhancement of upstream central carbon metabolism, i.e., uptake and metabolism of carbon sources, should be important so as to supply the precursor metabolites and coenzymes more efficiently.…”

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confidence: 99%

Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Hasegawa

Tanaka

Suda

et al. 2017

Appl Environ Microbiol

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In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly L-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. L-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc.IMPORTANCE Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.KEYWORDS Corynebacterium glutamicum, fructose metabolism, fructose-1-phosphate kinase, organic acid production, overexpression of glycolytic genes, regulation of glycolytic flux C orynebacterium glutamicum has been used as a powerful workhorse in the industrial production of amino acids, such as L-glutamate and L-lysine (1, 2). This organism behaves rather similarly to an aerobe because little growth is identified anaerobically without an alternative electron acceptor, nitrate (3, 4). In contrast, under oxygen-deprived conditions, C. glutamicum metabolizes glucose to L-lactate, succinate,

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“…Recent advances in metabolic engineering of S. cerevisiae have shown that systems and synthetic biology tools are advanced and readily available nowadays, allowing rational construction of capable production strains (Li & Borodina, 2014, Fletcher et al, 2015. Through combination of metabolic modelling and genetic engineering S. cerevisiae can be analysed and tailored in silico as well as in vivo , Borodina & Nielsen, 2014. This allows even very complex natural product biosynthetic pathways to be reconstructed in this organism (Galanie et al, 2015, Kayser & Averesch, 2015.…”

Section: Yeast In Biotechnologymentioning

confidence: 99%

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

Averesch¹

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Aromatic building blocks are amongst the most important bulk feedstocks in the chemical industry. As these compounds are commonly derived from petrochemistry, obtaining them is becoming more and more a matter of costs and sustainability. Biochemistry gives rise to a wealth of compounds that can potentially replace current petroleum-based chemicals or be used for novel materials. The aromatic compounds para-aminobenzoic acid (pABA) and para-hydroxybenzoic acid (pHBA) and the aromatics derived compound cis,cismuconic acid (ccMA) can be precursors for, but are not limited to, terephthalic or/and adipic acid. These are essential feedstocks for the production of PET and nylon. The three compounds can be derived from the shikimate pathway, an anabolic pathway leading to the biosynthesis of aromatic amino acids, present in certain prokaryotes and eukaryotes, including fungi. By combination of metabolic modelling with genetic engineering, a microbial production system based on the yeast Saccharomyces cerevisiae can be designed, which effectively channels flux into the target compounds.In order to develop a competitive bio-based process, yields, titers and rates need to be maximized. While productivity or rates in a process can be altered using genetic engineering, carbon yield, and pathway feasibility are stoichiometrically and thermodynamically predetermined. Both limitations need to be considered when designing a microbial production system. For formation of adipic acid and precursors many bio-based routes exists. More rational than just picking one for in vivo studies rather all available biochemical pathways were examined in silico using metabolic modelling. To compare theoretical yields and reaction thermodynamics an interface that allowed network-embedded thermodynamic analysis of elementary flux modes was developed. This allowed distinguishing between thermodynamically feasible and infeasible flux distributions. Feasible maximum theoretical product carbon yields were substantially different in E. coli and S. cerevisiae metabolic models and ranged from 32% to 92%. Further, many pathways appeared to be restricted by a thermodynamic equilibrium lying on the substrate side, some even infeasible.The only routes that deliver significant product yields and were thermodynamically favoured were shikimate pathway based. Being currently of strong scientific interest, recent implementations of these pathways in E. coli and S. cerevisiae were evaluated and strain construction strategies optimized, using the concept of constrained minimal cut-sets.Especially in S. cerevisiae a single non-obvious knock-out target allowed coupling of growth to product formation; in particular, the deletion of the pyruvate kinase reaction resulted in a minimum yield constraint of 28%.Though unique to shikimate pathway, this strategy is transferrable to other products which are derived from chorismate and also involve the formation of pyruvate as a by-product. This applies to pHBA. With further optimizations, the strategy was applied in...

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Advances in metabolic engineering of yeastSaccharomyces cerevisiaefor production of chemicals

Cited by 239 publications

References 74 publications

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

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