Metabolic Engineering of Pseudomonas putida KT2440 for De Novo Biosynthesis of Vanillic Acid.

Vanillic acid (VA), as a plant-derived phenolic acid compound, has widespread applications and good market prospects. However, the traditional production process cannot meet market demand. In this study, Pseudomonas putida KT2440 was used for de novo biosynthesis of VA. Multiple metabolic engineering strategies were applied to construct these P. putida-based cell factories, including the introduction of a Hs-OMTopt, engineering the cofactor S-adenosylmethionine supply pathway through the overexpression of metX and metH, reforming solubility of Hs-OMTopt, increasing a second copy of Hs-OMTopt, and the optimization of the fermentation medium. The resulting strain, XCS17, de novo biosynthesized 5.4 g/L VA from glucose in a fed-batch fermentation system; this is the highest VA production titer reported up to recently. This study showed that P. putida KT2440 is a robust platform for achieving the effective production of phenolic acids.

Metabolic engineering of Yarrowia lipolytica for high-level production of scutellarin.

Scutellarin drugs have been recognized as a key item in the national development of essential clinical emergency drugs for treating cardiovascular and cerebrovascular diseases; therefore, the market demand for scutellarin is growing rapidly. Microbial synthesis based on synthetic biology is a promising method for industrial production of scutellarin. In this study, the highest reported scutellarin titer in the shake flask of 703.01 ± 4.83 mg/L was achieved in Yarrowia lipolytica through the systematic metabolic engineering modifications, including screening for the optimal flavone-6-hydroxylase-cytochrome P450 reductase combination SbF6H-ATR2 to enhance P450 enzyme activity, increasing the copy numbers of rate-limiting enzyme genes, overexpressing ZWF1 and GND1 to increase NADPH supply, enhancing the supply of p-coumaric acid and uridine diphosphate glucose, and introducing the heterologous gene VHb to enhance oxygen supply. This study has significant implications for the industrial production of scutellarin and other valuable flavonoids in green economies.

Highly Efficient Biosynthesis of Protocatechuic Acid via Recombinant Pseudomonas putida KT2440.

Owing to their physiological activities, plant-derived phenolic acids, such as protocatechuic acid (PCA), have extensive applications and market prospects. However, traditional production processes present numerous challenges and cannot meet increasing market demands. Hence, we aimed to biosynthesize PCA by constructing an efficient microbial factory via metabolic engineering of Pseudomonas putida KT2440. Glucose metabolism was engineered by deleting the genes for gluconate 2-dehydrogenase to enhance PCA biosynthesis. To increase the biosynthetic metabolic flux, one extra copy of the genes aroGopt, aroQ, and aroB was inserted into the genome. The resultant strain, KGVA04, produced 7.2 g/L PCA. By inserting the degradation tags GSD and DAS to decrease the amount of shikimate dehydrogenase, PCA biosynthesis was increased to 13.2 g/L in shake-flask fermentation and 38.8 g/L in fed-batch fermentation. To the best of our knowledge, this was the first use of degradation tags to adjust the amount of a key enzyme at the protein level in P. putida KT2440, evidencing the remarkable potential of this method for naturally producing phenolic acids.

Metabolic engineering for the high-yield production of polydatin in Yarrowia lipolytica.

Polydatin, a glycosylated derivative of resveratrol, has better structural stability and biological activity than resveratrol. Polydatin is the extract of Polygonum cuspidatum, which has various pharmacological effects. Owing to its Crabtree-negative characteristics and high supply of malonyl-CoA, Yarrowia lipolytica was selected to produce polydatin. Initially, the resveratrol synthetic pathway was established in Y. lipolytica. By enhancing the shikimate pathway flow, redirecting carbon metabolism, and increasing the copies of key genes, a resveratrol yield of 487.77 mg/L was obtained. In addition, by blocking the degradation of polydatin, its accumulation was successfully achieved. Finally, by optimizing the glucose concentration and supplementing with two nutritional marker genes, a high polydatin yield of 6.88 g/L was obtained in Y. lipolytica, which is the highest titer of polydatin produced in a microbial host to date. Overall, this study demonstrates that Y. lipolytica has great potential for glycoside synthesis.

Construction of a p-coumaric and ferulic acid auto-regulatory system in Pseudomonas putida KT2440 for protocatechuate production from lignin-derived aromatics.

Lignin is a robust and underutilized aromatic heteropolymer on earth. The effective valorization of lignin into value-added products is an attractive research topic in lignocellulosic biorefineries. However, a low bioconversion rate, high process cost, low yield, and high toxicity of substrates hinder its further applications. In this study, an auto-regulatory system was developed and identified as an effective solution to diminish these challenging bottlenecks. First, a lignin-derived standard model aromatic p-coumaric acid (p-CA)-responsive biosensor was successfully developed through a series of rational engineering approaches in Pseudomonas putida KT2440. Furthermore, an auto-regulatory system was established, which rapidly coexpressed key rate-limiting enzymes, 4-hydroxybenzoate hydroxylase, vanillate-O-demethylase, and transporter HcnK under the biosensor element, to convert the mixture of p-CA and ferulic acid into a value-added platform chemical protocatechuate with a titer of 12.7 g/L. This study demonstrated that the constructed auto-regulatory platform is effective and economical for lignin valorization.

Metabolically engineering of Yarrowia lipolytica for the biosynthesis of naringenin from a mixture of glucose and xylose.

Xylose-inducible modules simultaneously expressing xylose utilization and naringenin biosynthesis pathways were developed in Yarrowia lipolytica to produce naringenin from a mixture of glucose and xylose. The naringenin synthetic pathway was constructed using a constitutive expression to yield 239.1 ± 5.1 mg/L naringenin. Furthermore, the introduction of an inducible pathway realized the dual function of xylose as a substrate and synthetic inducer, which coupled the xylose utilization with naringenin biosynthesis and increased production. Interestingly, the simultaneous enhancement of xylose reductase and xylose transporter expression along with that of xylitol dehydrogenase and xylulokinase can further improve the xylose utilization ability of Y. lipolytica. As expected, xylose-inducible synthesis of naringenin could achieved a titer of 715.3 ± 12.8 mg/L through the shake-flask cultivation level. Therefore, xylose-induced activation of both the xylose utilization and product biosynthesis pathway is considered to be an effective strategy for the biosynthesis of xylose-derived chemicals in yeast.

Engineering Prokaryotic Transcriptional Activator XylR as a Xylose-Inducible Biosensor for Transcription Activation in Yeast.

Biosensors regulated by specific substrates are needed to develop genetic tools to meet the needs of engineering microbial cell factories. Here, a xylose-inducible biosensor (xylbiosensor), comprising the Escherichia coli activation factor XylR, fusion activation domain (AD) VPRH, and a hybrid promoter with operator xylO, was established in Yarrowia lipolytica. The addition of xylose to an engineered Y. lipolytica strain harboring the xylbiosensor could trigger significant transcriptional activation of target genes, such as mcherry and the xylose utilization gene. Furthermore, a novel promoter Pleu-Pxo-Ptef was developed to construct a bidirectional expression system. The xylbiosensor showed good portability in Saccharomyces cerevisiae, suggesting its potential value in other eukaryotic cells. This study is the first to construct a "turn-on" xylbiosensor induced by xylose addition based on a prokaryotic activator XylR and eukaryotic universal AD. The xylbiosensor exhibits potential in pathway engineering for xylose utilization and xylose-derived product biosynthesis in yeast.

Engineering Yarrowia lipolytica for Enhanced Production of Arbutin.

Arbutin, a glycoside, is derived from the leaves of several plants, including wheat, pear, and bearberry plants, and has a significant role in the treatment of melanoma, cystitis, and cough. Here, we aimed to modify Yarrowia lipolytica to produce arbutin. To construct the arbutin synthetic pathway in Y. lipolytica, three genes (chorismate pyruvate-lyase (UbiC), 4-hydroxybenzoate 1-hydroxylase (MNX1), and hydroquinone glucosyltransferase (AS)) were codon-optimized and heterologously expressed. To maximize arbutin production, seven arbutin-biosynthesis molecular targets were overexpressed, and we found that the individual strengthening of DHS1 and DHS2 led to an 8.9- and 7.8-fold improvement in arbutin yield, respectively. Through optimization, a maximum arbutin titer of 8.6 ± 0.7 g/L was achieved using the finally engineered strain, po1f-At09. Overall, this is the first report of heterologous arbutin synthesis in Y. lipolytica at a high titer. Furthermore, this work opens a possibility for the overproduction of shikimate pathway derivatives in Y. lipolytica.