Metabolic and transport engineering enable efficient de novo biosynthesis of patchoulol in Saccharomyces cerevisiae.

Enhanced isoprene biosynthesis in Saccharomyces cerevisiae by engineering of the native acetyl-CoA and mevalonic acid pathways with a push-pull-restrain strategy

Terpenoids are a diverse class of compounds with significant application potential. While prokaryotic bacteria synthesize terpenoids via the methylerythritol-4-phosphate (MEP) pathway, fungi utilize the mevalonate (MVA) pathway. The MVA pathway has been widely employed for efficient terpenoid production in bacteria such as Escherichia coli, but the MEP pathway performs poorly for biosynthesis in yeast. In this study, we constructed a compartmentalized MEP pathway to enhance monoterpenoid production in Saccharomyces cerevisiae. By introducing a geraniol synthase, we initially achieved the production of geraniol from glucose. Further effective incorporation of a cytosolic MEP pathway with nine enzymes increased geraniol production by 174.5%. However, this also significantly inhibited cell growth. Overexpression analysis revealed that flavodoxin and flavodoxin reductase were major contributors to growth inhibition, which could also be a factor limiting the application of the MEP pathway. To address these issues, we employed peroxisomal compartmentalization to isolate the MEP pathway from cytosolic metabolism. This strategy alleviated growth inhibition and improved geraniol production by 93.18% compared to that of cytosolic expression. Through additional metabolic engineering, we optimized peroxisomal geraniol production, achieving a yield of 30.64 mg/L. Our findings demonstrate the potential of compartmentalized MEP pathway expression as a viable approach for enhancing terpenoid biosynthesis in yeast, offering valuable insights for future metabolic engineering efforts.

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.

Menthol, a high-value commodity monoterpenoid chemical, holds an important market share commercially because of its distinct functions. The menthol on the market mainly originates from plant extraction, which is facing challenges such as the seasonal fluctuations and long growth cycle of plants. Therefore, this study attempted to realize the de novo synthesis of menthol through microbial fermentation. First, through heterologous expression and subcellular localization observation, a synthetic route from glucose to (−)-menthol was successfully designed and constructed in Saccharomyces cerevisiae. Then, the mevalonate (MVA) pathway was enhanced, and the expression of farnesyl diphosphate synthase (ERG20) was dynamically regulated to improve the synthesis of D-limonene, a key precursor of (−)-menthol. Shake flask fermentation results showed that the D-limonene titer of the recombinant strain reached 459.59 mg/L. Next, the synthesis pathway from D-limonene to (−)-menthol was strengthened, and the fermentation medium was optimized. The (−)-menthol titer of 6.28 mg/L was obtained, implying that the de novo synthesis of menthol was successfully realized for the first time. This study provides a good foundation for the synthesis of menthol through microbial fermentation.

Squalene, a linear triterpene compound characterized by its distinctive isoprenoid backbone with six transisoprene units, is widely used in the medicinal, nutraceutical, and cosmetic industries. The escalating global demand for squalene, coupled with growing ethical concerns over shark-derived sources and the inherent limitations of plant extraction (low yield) and chemical synthesis (environmental burden), has propelled microbial biosynthesis as a sustainable alternative. While substantial progress has been made in elucidating the mevalonate pathway and regulatory mechanisms of squalene biosynthesis, achieving industrially viable titers through microbial platforms remains an unresolved challenge. This review systematically summarizes recent advances in squalene biosynthesis using yeast chassis, with a focus on metabolic engineering strategies implemented in Saccharomyces cerevisiae and Yarrowia lipolytica. Furthermore, we elaborated on how squalene yields a diverse array of downstream derivatives through intricate enzymatic reactions. These derivatives—including triterpenoid saponins, triterpenoid acids, and steroids—exhibit significant applications in the pharmaceutical, nutraceutical, and cosmetic sectors. By integrating systems metabolic engineering with emerging synthetic biology tools, this work provides a roadmap for advancing strain engineering toward economically feasible squalene biomanufacturing.

Panax ginseng Meyer is one of the most important medicinal plants in Asia, and ginseng has attracted considerable attention worldwide. Triterpene saponins (ginsenosides) are the main bioactive compounds in P. ginseng. The isoprene units of triterpene are derived from the mevalonic acid (MVA) pathway. We cloned four genes involved in MVA pathway using rapid amplification of cDNA ends by polymerase chain reaction. Additionally, we investigated the transcript levels of 11 genes involved in the terpenoid pathway in different organs and cell suspension cultures of P. ginseng. The full-length cDNA sequences were as follows: PgHMGS (1764 bp; 1407-bp ORF), PgHMGR (1992 bp; 1722-bp ORF), PgPMK (2170 bp; 1530-bp ORF), and PgMVD (1759 bp; 1263-bp ORF). The highest expression level of all genes was found in fine roots. The total ginsenoside contents in different organs were ranked in the following descending order: leaf > fine root > lateral root > red berry > main root > petiole > stem. Campesterol and stigmasterol were detected in all organs but at different concentrations. The total phytosterol content was highest in fine root (147.8 μg/100 mg dry weight (DW)), and was lowest in the stem (86.4 μg/100 mg DW). Four enzymes in the MVA pathway were cloned and characterized in P. ginseng. Such genes play important roles in terpenoid biosynthesis and may have applications in the metabolic engineering of ginsenoside production.

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker’s yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

The introduction of heterologous pathways into microorganisms for high‐level synthesis of valuable compounds would be extremely meaningful. However, the endogenous heterologous pathway conversion point frequently serves as a bottleneck that limits product synthesis. (+)‐Nootkatone is an important functional sesquiterpenoid with a unique grapefruit flavor, which is frequently used as a fragrance. The low catalytic efficiency of the valencene synthase CnVS, the first heterologous enzyme in the biosynthesis of (+)‐nootkatone, is the major challenge that restricts the production of (+)‐nootkatone in Saccharomyces cerevisiae. In this study, we modified CnVS on the basis of the catalytic environment and mechanism of the enzyme. The residue M560 involving in alkyl transfer during substrate cyclization was found to affect activity. By tuning the residue, mutant M560L exhibited a 60% increase in the unit cell yield of (+)‐valencene, which is the precursor of (+)‐nootkatone. Furthermore, multistep strategies of metabolic engineering were employed that transferred the metabolic flux to the target product synthesis pathway and displayed the superimposed effect of metabolic and protein engineering on (+)‐nootkatone yield. Finally, multicopy integration of tHMG1 and ERG20ERG20‐GSG‐CnVSM560L boosted the production of (+)‐nootkatone to 85.43 mg/L in flask and 804.96 mg/L (5.29 mg/[L h]) in bioreactor. In conclusion, this study presents a paradigm for constructing microbial cell factories using a combination of protein and metabolic engineering in S. cerevisiae.

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

BackgroundXylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates.ResultsIn this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae, were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha.ConclusionsThe O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

Patchoulol is a natural sesquiterpene alcohol with extensive applications in cosmetics and pharmaceuticals. In this study, we first constructed the synthesis pathway of patchoulol in Saccharomyces cerevisiae by expressing the patchoulol synthase PTS gene using the strong promoter GAL1. Afterward, the metabolic flux of the precursor was enhanced by strengthening the mevalonate pathway and balancing the precursor competition pathway, resulting in a 32.74-fold increase in patchoulol production. Subsequently, the supply of acetyl-CoA in yeast was increased by modifying transcriptional regulators and modulating the acetyl-CoA pathway, and the titer of patchoulol reached 155.94 mg/L. Finally, optimization of the fermentation conditions resulted in a titer of 195.96 mg/L in the shake flasks. Further, batch-fed fermentation in a 5 L bioreactor yielded 1.95 g/L. This work accelerated the development of a microbial cell factory for the production of patchoulol.

Microbial fermentation is an important technology for the conversion of renewable resources to chemicals. In this paper, we describe the application of metabolic engineering for the development of two new fermentation processes: the microbial conversion of sugars to 1,3-propanediol (1,3-PD) and 1,2-propanediol (1,2-PD). A variety of naturally occurring organisms ferment glycerol to 1,3-PD, but no natural organisms ferment sugars directly to 1,3-PD. We first describe the fed-batch fermentation of glycerol to 1,3-PD by Klebsiella pneumoniae. We then present various approaches for the conversion of sugars to 1,3-PD, including mixed-culture fermentation, cofermentation of glycerol and glucose, and metabolic engineering of a "sugars to 1,3-PD" pathway in a single organism. Initial results are reported for the expression of genes from the K. pneumoniae 1,3-PD pathway in Saccharomyces cerevisiae. The best naturally occurring organism for the fermentation of sugars to 1,2-PD is Thermoanaerobacterium thermosaccharolyticum. We describe the fermentation of several different sugars to 1,2-PD by this organism in batch and continuous culture. We report that Escherichia coli strains engineered to express either aldose reductase or glycerol dehydrogenase convert glucose to (R)-1,2-PD. We then analyze the ultimate potential of fermentation processes for the production of propanediols. Linear optimization studies indicate that, under aerobic conditions, propanediol yields that approach the theoretical maximum are possible and CO2 is the primary coproduct. Without the need to produce acetate, final product titers in the range of 100 g/L should be possible; the high titers and low coproduct levels should make product recovery and purification straightforward. The examples given in this paper illustrate the importance of metabolic engineering for fermentation process development in general.

SummaryAdvances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by‐passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha‐4,11‐diene after cultivation with [4‐13C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.