RESUMO
The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The ß-carotene 15,15'-dioxygenase (BCO) cleaves ß-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated ß-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous ß-carotene biosynthetic genes, GGS1F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the ß-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in ß-carotene level, but less than 5% of the consumed ß-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.
Assuntos
Yarrowia , Antioxidantes , Hidroxitolueno Butilado/metabolismo , Detergentes/metabolismo , Polissorbatos/metabolismo , Vitamina A/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , beta Caroteno/metabolismoRESUMO
The endoplasmic reticulum (ER) is a dynamic organelle that synthesizes and folds proteins. An imbalance between the ER protein synthesis load and its folding capacity triggers the unfolded protein response, thereby restoring normal ER functions via size adjustment. Inspired by such inherent genetic programming events, we engineered Saccharomyces cerevisiae to expand the ER by overexpressing a key ER size regulatory factor, INO2. ER space expansion enhanced ER protein synthesis and folding capacity, and relieved metabolic constraints imposed by the limited enzyme abundance. Harnessing the yeast ER for metabolic engineering, we ultimately increased the production of squalene and cytochrome P450-mediated protopanaxadiol by 71-fold and 8-fold, compared to their respective control strains without overexpression of INO2. Furthermore, genome-wide transcriptome analysis of the ER-expanded strain revealed that the significant improvement in terpene production was associated with global rewiring of the metabolic network. Therefore, the yeast ER can be engineered as a specialized compartment for enhancing terpene production, representing new possibilities for the high-level production of other value-added chemicals.
Assuntos
Retículo Endoplasmático , Engenharia Metabólica , Saccharomyces cerevisiae , Terpenos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.
Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Hidrogênio/química , Engenharia Metabólica/métodos , Modelos Químicos , Amido/química , Água/química , Oxirredução , Proteínas Recombinantes/químicaRESUMO
Sugar phosphates cannot be produced easily by microbial fermentation because negatively-charged compounds cannot be secreted across intact cell membrane. D-xylulose 5-phosphate (Xu5P), a very expensive sugar phosphate, was synthesized from D-xylose and polyphosphate catalyzed by enzyme cascades in one pot. The synthetic enzymatic pathway comprised of xylose isomerase and xylulokinase was designed to produce Xu5P, along with a third enzyme, polyphosphate kinase, responsible for in site ATP regeneration. Due to the promiscuous activity of the ATP-based xylulokinase from a hyperthermophilic bacterium Thermotoga maritima on polyphosphate, the number of enzymes in the pathway was minimized to two without polyphosphate kinase. The reactions catalyzed by the two-enzyme and three-enzyme pathways were compared for Xu5P production, and the reaction conditions were optimized by examining effects of reaction temperature, enzyme ratio and substrate concentration. The optimized two-enzyme system produced 32 mM Xu5P from 50 mM xylose and polyphosphate after 36 h at 45°C. Biosynthesis of less costly Xu5P from D-xylose and polyphosphate could be highly feasible via this minimized two-enzyme pathway.
Assuntos
Enzimas/metabolismo , Pentosefosfatos/biossíntese , Polifosfatos/metabolismo , Xilose/metabolismo , Trifosfato de Adenosina/metabolismo , Aldose-Cetose Isomerases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Temperatura , Thermotoga maritima/enzimologiaRESUMO
Xylulokinase (XK, E.C. 2.7.1.17) is one of the key enzymes in xylose metabolism and it is essential for the activation of pentoses for the sustainable production of biocommodities from biomass sugars. The open reading frame (TM0116) from the hyperthermophilic bacterium Thermotoga maritima MSB8 encoding a putative xylulokinase were cloned and expressed in Escherichia coli BL21 Star (DE3) in the Luria-Bertani and auto-inducing high-cell-density media. The basic biochemical properties of this thermophilic XK were characterized. This XK has the optimal temperature of 85 °C. Under a suboptimal condition of 60 °C, the k cat was 83 s⻹, and the K(m) values for xylulose and ATP were 1.24 and 0.71 mM, respectively. We hypothesized that this XK could work on polyphosphate possibly because this ancestral thermophilic microorganism utilizes polyphosphate to regulate the Embden-Meyerhof pathway and its substrate-binding residues are somewhat similar to those of other ATP/polyphosphate-dependent kinases. This XK was found to work on low-cost polyphosphate, exhibiting 41 % of its specific activity on ATP. This first ATP/polyphosphate XK could have a great potential for xylose utilization in thermophilic ethanol-producing microorganisms and cell-free biosystems for low-cost biomanufacturing without the use of ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polifosfatos/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Animais , Gatos , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glicólise , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Temperatura , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Xilose/metabolismo , Xilulose/metabolismoRESUMO
The supply of terpenes is often limited by their low extraction yield from natural resources, such as plants. Thus, microbial biosynthesis has emerged as an attractive platform for the production of terpenes. Many strategies have been applied to engineer microbes to improve terpene production capabilities; however, functional expression of heterologous proteins such as cytochrome P450 enzymes (P450s) in microbes is a major obstacle. This study reports the successful pairing of cognate chaperones and P450s for functional heterologous expression in Saccharomyces cerevisiae. This chaperone pairing was exploited to facilitate the functional assembly of the protopanaxadiol (PPD) biosynthesis pathway, which consists of a P450 oxygenase and a P450 reductase redox partner originating from Panax ginseng and Arabidopsis thaliana, respectively. We identified several chaperones required for protein folding in P. ginseng and A. thaliana and evaluated the impact of the coexpression of the corresponding chaperones on the synthesis and activity of PPD biosynthesis enzymes. Expression of a chaperone from P. ginseng (PgCPR5), a cognate of PPD biosynthesis enzymes, significantly increased PPD production by more than 2.5-fold compared with that in the corresponding control strain. Thus, pairing of chaperones with heterologous enzymes provides an effective strategy for the construction of challenging biosynthesis pathways in yeast.
Assuntos
Sistema Enzimático do Citocromo P-450 , Saccharomyces cerevisiae , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismoRESUMO
BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.
RESUMO
Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for ß-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.
Assuntos
Saccharomyces cerevisiae , Terpenos , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo , Terpenos/metabolismo , beta Caroteno/metabolismoRESUMO
Microbial production of many lipophilic compounds is often limited by product toxicity to host cells. Engineering cell walls can help mitigate the damage caused by lipophilic compounds by increasing tolerance to those compounds. To determine if the cell wall engineering would be effective in enhancing lipophilic compound production, we used a previously constructed squalene-overproducing yeast strain (SQ) that produces over 600 mg/L of squalene, a model membrane-damaging lipophilic compound. This SQ strain had significantly decreased membrane rigidity, leading to increased cell lysis during fermentation. The SQ strain was engineered to restore membrane rigidity by activating the cell wall integrity (CWI) pathway, thereby further enhancing its squalene production efficiency. Maintenance of CWI was associated with improved squalene production, as shown by cell wall remodeling through regulation of Ecm33, a key regulator of the CWI pathway. Deletion of ECM33 in the SQ strain helped restore membrane rigidity and improve stress tolerance. Moreover, ECM33 deletion suppressed cell lysis and increased squalene production by approximately 12% compared to that by the parent SQ strain. Thus, this study shows that engineering of the yeast cell wall is a promising strategy for enhancing the physiological functions of industrial strains for production of lipophilic compounds.
Assuntos
Parede Celular/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo , Parede Celular/genética , Fermentação , Deleção de Genes , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ginseng (Panax ginseng) and its bioactive components, ginsenosides, are popular medicinal herbal products, exhibiting various pharmacological effects. Despite their advocated use for medication, the long cultivation periods of ginseng roots and their low ginsenoside content prevent mass production of this compound. Yeast Saccharomyces cerevisiae was engineered for production of protopanaxadiol (PPD), a type of aglycone characterizing ginsenoside. PPD-producing yeast cell factory was further engineered by obtaining a balance between enzyme expressions and altering cofactor availability. Different combinations of promoters (PGPD, PCCW12, and PADH2) were utilized to construct the PPD biosynthetic pathway. Rerouting the redox metabolism to improve NADPH availability in the engineered S. cerevisiae also increased PPD production. Combining these approaches resulted in more than an 11-fold increase in PPD titer over the initially constructed strain. The series of metabolic engineering strategies of this study provides a feasible approach for the microbial production of PPD and development of microbial platforms producing other industrially-relevant terpenoids.
Assuntos
Vias Biossintéticas , NADP/biossíntese , Saccharomyces cerevisiae/metabolismo , Sapogeninas/metabolismo , Carbono/farmacologia , Engenharia Metabólica , Oxirredução , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sapogeninas/químicaRESUMO
A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.
Assuntos
DNA/genética , Escherichia coli/genética , Biblioteca Gênica , Mutagênese , Clonagem Molecular , Primers do DNA/metabolismo , Evolução Molecular Direcionada , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/genética , Transformação Bacteriana , Proteína Vermelha FluorescenteRESUMO
Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.