Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.

Recent advances in the microbial production of squalene.

Squalene is a triterpene hydrocarbon, a biochemical precursor for all steroids in plants and animals. It is a principal component of human surface lipids, in particular of sebum. Squalene has several applications in the food, pharmaceutical, and medical sectors. It is essentially used as a dietary supplement, vaccine adjuvant, moisturizer, cardio-protective agent, anti-tumor agent and natural antioxidant. With the increased demand for squalene along with regulations on shark-derived squalene, there is a need to find alternatives for squalene production which are low-cost as well as sustainable. Microbial platforms are being considered as a potential option to meet such challenges. Considerable progress has been made using both wild-type and engineered microbial strains for improved productivity and yields of squalene. Native strains for squalene production are usually limited by low growth rates and lesser titers. Metabolic engineering, which is a rational strain engineering tool, has enabled the development of microbial strains such as Saccharomyces cerevisiae and Yarrowia lipolytica, to overproduce the squalene in high titers. This review focuses on key strain engineering strategies involving both in-silico and in-vitro techniques. Emphasis is made on gene manipulations for improved precursor pool, enzyme modifications, cofactor regeneration, up-regulation of limiting reactions, and downregulation of competing reactions during squalene production. Process strategies and challenges related to both upstream and downstream during mass cultivation are detailed.

Adaptive evolution of engineered yeast for squalene production improvement and its genome-wide analysis.

In the present study, the adaptive evolution of a metabolically engineered Saccharomyces cerevisiae strain in the presence of an enzyme inhibitor terbinafine for enhanced squalene accumulation via serial transfer leads to the development of robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production efficiency was identified at a specific growth rate of 0.28 h-1 with a final squalene titer of 193 mg/L, which is 16.5-fold higher than the BY4741 and 3-fold higher over the metabolically engineered SK22 strain. Whole-genome sequencing comparison between the reference strain and the evolved variant SK23 has led to the identification of 462 single-nucleotide variants (SNVs) between both strains, with 102 SNVs affecting metabolism-related genes. It was also established that F420I mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant strains was investigated by fluorescent techniques proving that the neutral lipid content and clustering of lipid droplets increase with an increase in squalene.

The biology of ergothioneine, an antioxidant nutraceutical.

Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

Progress in terpene synthesis strategies through engineering of Saccharomyces cerevisiae.

Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses' viz., Escherichia coli and Saccharomyces cerevisiae have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40 g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer S. cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S. cerevisiae genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.

In silico target-based strain engineering of Saccharomyces cerevisiae for terpene precursor improvement.

Systems-based metabolic engineering enables cells to enhance product formation by predicting gene knockout and overexpression targets using modeling tools. FOCuS, a novel metaheuristic tool, was used to predict flux improvement targets in terpenoid pathway using the genome-scale model of Saccharomyces cerevisiae, iMM904. Some of the key knockout target predicted includes LYS1, GAP1, AAT1, AAT2, TH17, KGD-m, MET14, PDC1 and ACO1. It was also observed that the knockout reactions belonged either to fatty acid biosynthesis, amino acid synthesis pathways or nucleotide biosynthesis pathways. Similarly, overexpression targets such as PFK1, FBA1, ZWF1, TDH1, PYC1, ALD6, TPI1, PDX1 and ENO1 were established using three different existing gene amplification algorithms. Most of the overexpression targets belonged to glycolytic and pentose phosphate pathways. Each of these targets had plausible role for improving flux toward sterol pathway and were seemingly not artifacts. Moreover, an in vitro study as validation was carried with overexpression of ALD6 and TPI1. It was found that there was an increase in squalene synthesis by 2.23- and 4.24- folds, respectively, when compared with control. In general, the rationale for predicting these in silico targets was attributed to either increasing the acetyl-CoA precursor pool or regeneration of NADPH, which increase the sterol pathway flux.

Studies on Squalene Biosynthesis and the Standardization of Its Extraction Methodology from Saccharomyces cerevisiae.

In this study, a homogenization-based extraction method was developed and was compared to five conventional methods of squalene extraction. Squalene recovered from this novel procedure gave 3.5-fold, 10-fold, 16-fold, and 8.1-fold higher yield than standard procedures, viz., saponification with 60% KOH, acidic saponification, saponification with 18% KOH, and glass beads method, respectively. Furthermore, this procedure has been evaluated on laboratory Saccharomyces cerevisiae strains such as BY4742 and CEN.PK2-1C (native), deletion strains (ERG6 and ERG11), and tHMG1 overexpressed S. cerevisiae strains. When sonication method of cell lysis was replaced with homogenization, it was found that the yields were significantly higher and reached a value of 9 mg/g DCW in case of BY4742. In addition, squalene yield in ergosterol mutant strains has been analyzed and was found to be 1.8-fold and 3.4-fold higher in ERG6 and ERG11 deletion strains, respectively, than in BY4742. Squalene was also found to be higher at the optimized temperature of 30 °C and pH 6.0. Furthermore, tolerance of S. cerevisiae to external squalene at various concentrations has been carried and found that the organism was tolerant up to 25 g/L of squalene.

Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae.

Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes', viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.