RESUMEN
Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid-a crucial precursor to artemisinin-is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.
Asunto(s)
Productos Biológicos , Terpenos , FermentaciónRESUMEN
BACKGROUND: Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites. RESULTS: In this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W-N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W-N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation. CONCLUSIONS: The results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.
Asunto(s)
Ácido Mevalónico , Saccharomyces cerevisiae , Monoterpenos Acíclicos , Carbono/metabolismo , Variaciones en el Número de Copia de ADN , Difosfatos/metabolismo , Geraniltranstransferasa/genética , Geraniltranstransferasa/metabolismo , Hemiterpenos , Ingeniería Metabólica/métodos , Ácido Mevalónico/metabolismo , Monoterpenos/metabolismo , Compuestos Organofosforados , Pentanoles , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.
Asunto(s)
Monoterpenos Acíclicos/química , Monoterpenos Acíclicos/metabolismo , Ácido Glutámico/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Synechocystis/metabolismo , Escherichia coli/genética , Aromatizantes/química , Aromatizantes/metabolismo , Ingeniería Genética , Ácido Glutámico/química , Estereoisomerismo , Synechocystis/genéticaRESUMEN
Identification of diterpene synthase-encoding genes together with synthetic biology technology offers an opportunity for the biosynthesis of cis-abienol. The methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways were both engineered for cis-abienol production in Escherichia coli, which improved the cis-abienol yield by approximately 7-fold and 31-fold, respectively, compared to the yield obtained by overexpression of the MEP pathway alone or the original MEP pathway. Furthermore, systematic optimization of cis-abienol biosynthesis was performed, such as diterpene synthase screening and two-phase cultivation. The combination of bifunctional class I/II cis-abienol synthase from Abies balsamea ( AbCAS) and class II abienol synthase from Salvia sclarea ( SsTPS2) was found to be the most effective. By using isopropyl myristate as a solvent in two-phase cultivation, cis-abienol production reached 634.7 mg/L in a fed-batch bioreactor. This work shows the possibility of E. coli utilizing glucose as a carbon source for cis-abienol biosynthesis through a modified pathway.
Asunto(s)
Diterpenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica , Ácido Mevalónico/metabolismo , Naftoles/metabolismo , Abies/enzimología , Vías Biosintéticas , Diterpenos/química , Fermentación , Glucosa/metabolismo , Naftoles/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Salvia/enzimologíaRESUMEN
Methyl anthranilate (MANT) is a widely used compound to give grape scent and flavor, but is currently produced by petroleum-based processes. Here, we report the direct fermentative production of MANT from glucose by metabolically engineered Escherichia coli and Corynebacterium glutamicum strains harboring a synthetic plant-derived metabolic pathway. Optimizing the key enzyme anthranilic acid (ANT) methyltransferase1 (AAMT1) expression, increasing the direct precursor ANT supply, and enhancing the intracellular availability and salvage of the cofactor S-adenosyl-l-methionine required by AAMT1, results in improved MANT production in both engineered microorganisms. Furthermore, in situ two-phase extractive fermentation using tributyrin as an extractant is developed to overcome MANT toxicity. Fed-batch cultures of the final engineered E. coli and C. glutamicum strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT.
Asunto(s)
Corynebacterium glutamicum , Escherichia coli , Aromatizantes/metabolismo , Ingeniería Metabólica/métodos , ortoaminobenzoatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos/microbiología , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Redes y Vías Metabólicas , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Brown seaweed contains up to 67% of carbohydrates by dry weight and presents high potential as a polysaccharide feedstock for biofuel production. To effectively use brown seaweed as a biomass, degradation of alginate is the major challenge due to its complicated structure and low solubility in water. This study focuses on the isolation of alginate degrading bacteria, determining of the optimum fermentation conditions, as well as comparing the conventional single fermentation system with the two-phase fermentation system which is separately using alginate and mannitol extracted from Laminaria japonica. Maximum yield of organic acids production and volatile solids reduction obtained were 0.516 g/g and 79.7%, respectively, using the two-phase fermentation system in which alginate fermentation was carried out at pH 7 and mannitol fermentation at pH 8. The two-phase fermentation system increased the yield of organic acids production by 1.14 times and led to a 1.45-times reduction of VS when compared to the conventional single fermentation system at pH 8. The results show that the two-phase fermentation system improved the utilization of alginate by separating alginate from mannitol leading to enhanced alginate lyase activity.