Engineering the Activity of a Newly Identified Arylalkylamine N-Acetyltransferase in the Acetylation of 5-Hydroxytryptamine.

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole-cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5-HT into NAS. Additionally, its catalytic efficiency (kcat/Km) was enhanced by 2-fold while retaining the thermostability (Tm>45 °C). In the up-scaled reaction with a substrate loading of 50 mM, the whole-cell system incorporating variant M3 achieved a 99 % conversion of 5-HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach.

BACKGROUND: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and ß-amyrin on simple sugar and waste cooking oil. RESULTS: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L ß-amyrin. CONCLUSIONS: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

The association of plasma peroxiredoxin 3 with insulin in pregnant women.

Peroxiredoxin 3 (PRX3) is predominantly located in mitochondria and plays a major role in scavenging hydrogen peroxide of mitochondria. In the present study, we detected plasma PRX3 in pregnant women receiving oral glucose tolerance test at 24-28 gestational weeks. Plasma PRX3 was significantly increased about 1 h later than insulin secretion. In vitro detection of PRX3 in mouse islet cells showed up-regulation by more than 2-fold at 1 h and reached its top at 2 h of glucose stimulation, and the PRX3 level in cultured mediums was concomitantly elevated in a glucose concentration-dependent manner. In addition, both fasting plasma insulin and PRX3 were significantly higher in the subjects of term pregnancy as compared to that at 24-28 gestational weeks, and there was a positive correlation between plasma PRX3 and insulin. Our results indicate that PRX3 plays an active role in the response to insulin release. The positive association of plasma PRX3 and insulin suggest PRX3 to be a potential indicator of high insulin resistance.

Plasma level of peroxiredoxin 3 in patients with polycystic ovarian syndrome.

BACKGROUND: As a member of peroxiredoxin (PRX) family, PRX3 is predominantly located in mitochondria and plays an important role of free radical scavenging. Since a body of evidence demonstrated the involvement of PRX3 in insulin secretion, insulin sensitivity, and glucose metabolism, the present study was conducted to investigate the role of PRX3 in the pathogenesis of polycystic ovarian syndrome (PCOS) featured in insulin resistance. METHODS: Enzyme-linked immunosorbent assay was performed to detect plasma PRX3 in PCOS patients and control subjects. Levels of reactive oxygen species (ROS) and oxidized PRXs were detected in mouse islet cells treated with gradient glucose. RESULTS: We did not find significant difference of fasting plasma PRX3 between PCOS patients and controls. No association was noticed between fasting plasma PRX3 and fasting plasma glucose or insulin. After oral glucose tolerance test (OGTT), PCOS patients showed higher levels of both glucose and insulin as compared to controls. The plasma level of PRX3 was significantly increased at 2 h and began to fall back at 3 h of OGTT. There was a one-hour time lag of peak values between plasma PRX3 and insulin, and the plasma PRX3 at 2 h was positively correlated with the insulin level at 1 h of OGTT of PCOS patients. In addition, the level of ROS was significantly elevated at 1 h and oxidized PRX3 was increased dramatically at 2 h of 16.7mM glucose stimulation in mouse islet cells. CONCLUSION: It seems that PRX3 does not show its antioxidant function under baseline conditions. Instead, PRX3 responds to oxidative stress induced by rapid increase of insulin and glucose in patients with PCOS.

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

BACKGROUND: Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. RESULTS: The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. CONCLUSIONS: These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

The Potential Role of Probiotics in Cancer Prevention and Treatment.

The human gut microbiota has a significant effect on many aspects of human physiology such as metabolism, nutrient absorption, and immune function. Imbalance of the microbiota has been implicated in many disorders including inflammatory bowel disease, obesity, asthma, psychiatric illnesses, and cancers. As a kind of functional foods, probiotics have been shown to play a protective role against cancer development in animal models. Clinical application of probiotics indicated that some probiotic strains could diminish the incidence of postoperative inflammation in cancer patients. Chemotherapy or radiotherapy-related diarrhea was relieved in patients who were administered oral probiotics. The present review summarizes the up-to-date studies on probiotic effects and the underlying mechanisms related to cancer. At present, it is commonly accepted that most commercial probiotic products are generally safe and can improve the health of the host. By modulating intestinal microbiota and immune response, some strains of probiotics can be used as an adjuvant for cancer prevention or/and treatment.

Sustainable biosynthesis of squalene from waste cooking oil by the yeast Yarrowia lipolytica.

Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering principles to enhance squalene production in the nonconventional yeast Yarrowia lipolytica, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway - specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase - we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using Y. lipolytica cell factories, contributing to circular economy initiatives by valorizing waste materials.

Identification and characterization of a novel C20-elongase gene from the marine microalgae, Pavlova viridis, and its use for the reconstitution of two pathways of long-chain polyunsatured fatty acids biosynthesis in Saccharomyces cerevisiae.

The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway.

Combining Metabolic Engineering and Lipid Droplet Storage Engineering for Improved α-Bisabolene Production in Yarrowia Lipolytica.

Bisabolene is a bioactive sesquiterpene with a wide range of applications in food, cosmetics, medicine, and aviation fuels. Microbial production offers a green, efficient, and sustainable alternative. In this study, we focused on improving the titers of α-bisabolene in Yarrowia lipolytica by applying two strategies, (i) optimizing the metabolic flux of α-bisabolene biosynthetic pathway and (ii) sequestering α-bisabolene in lipid droplet, thus alleviating its inherent toxicity to host cells. We showed that overexpression of DGA1 and OLE1 to increase lipid content and unsaturated fatty acid levels was essential for boosting the α-bisabolene synthesis when supplemented with auxiliary carbon sources. The final engineered strain Po1gαB10 produced 1954.3 mg/L α-bisabolene from the waste cooking oil under shake flask fermentation, which was 96-fold higher than the control strain Po1gαB0. At the time of writing, our study represents the highest reported α-bisabolene titer in the engineered Y. lipolytica cell factory. This work describes novel strategies to improve the bioproduction of α-bisabolene that potentially may be applicable for other high-value terpene products.

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences.

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆(12)-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆(15)-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆(9)-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Identification of a novel C22-∆4-producing docosahexaenoic acid (DHA) specific polyunsaturated fatty acid desaturase gene from Isochrysis galbana and its expression in Saccharomyces cerevisiae.

Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-∆4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ∆4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomyces cerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ∆4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-∆4-PUFAs-specific desaturase activity.

[Effects of low temperature and exogenous unsaturated fatty acids on fatty acid desaturase gene expression of Mortierella alpina ATCC 16266].

OBJECTIVE: In order to investigate the regulatory mechanisms of Mortierella alpina desaturase genes by low temperature and exogenous unsaturated fatty acids (UFAs) at the transcriptional level. METHODS: We performed time-course studies of fatty acid desaturase gene expression by real-time PCR and determined fatty acid desaturase gene promoter activity using promoter-reporter constructs. RESULTS: Relative expression results in real-time PCR showed that the mRNA levels of three fatty acid desaturase genes (Fad6, Fad12 and Fad3) were rapidly and transiently enhanced after 1 h of shifting to low temperature, in contrast, high concentration of exogenous oleic acid (OA) which was a monounsaturated fatty acid containing a D9 double bond suppressed the transcription of these genes and the transcriptional response appeared to be rapid and transient. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. The pFAD6 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by addition of UFAs to the media, and alpha-linolenic acid (ALA) containing three double bonds appeared to have a more effective inhibition than linoleic acid (LA) and OA. CONCLUSION: These results indicate that there may be post-transcriptional control and other modes of regulation of UFAs synthesis in M. alpina when facing different stimuli such as low temperature and exogenous unsaturated fatty acids besides the regulation in the transcription of fatty acid desaturase genes at the initial stage. Also, there may be an unknown end-product (changes in fatty acid compositions) feedback regulation in the transcription of fatty acid desaturase genes to maintain cellular UFAs' homeostasis. In a word, we assessed mechanisms of transcriptional regulation in M. alpina fatty acid desaturase gene expression for the first time and we wish to make it possible to obtain a better understanding of the mechanisms and get some theoretical knowledge to offer some guidance to the industrial production of UFAs by transgenic technology and microbial fermentation technology.

[Production of carboxylic acids by metabolically engineered Yarrowia lipolytica: a review].

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.

Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil.

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

Effects of different carbon sources on the growth, fatty acids production, and expression of three desaturase genes of Mortierella alpina ATCC 16266.

On the molecular and biochemical levels, the effects of different carbon sources on biomass production, fatty acid biosynthesis, and gene expression of three desaturases were investigated in Mortierella alpina ATCC 16266, at a stationary phase, which is an important filamentous fungus capable of producing various polyunsaturated fatty acids (PUFAs). The maximum mycelial biomass was achieved using sucrose as carbon source. However, the highest productivity of total lipids was shown to be no biomass associated. In addition, glucose was the preferred carbon source for the expression of three desaturase genes compared to others, but the change at the corresponding fatty acid product's level of these desaturase genes was not in accordance with the change measured at the mRNA level among those carbon sources that we utilized. Significant discrepancies between the mRNA expression and the product abundance may indicate post-transcriptional regulatory mechanisms of these desaturases.

[Advances in adaptive laboratory evolutionary engineering to microbial breeding].

In recent years, adaptive laboratory evolution (ALE) has emerged as a powerful tool for basic research in microbiology (e.g., molecular mechanisms of microbial evolution) and efforts on evolutionary engineering of microbial strains (e.g., accelerated evolution of industrial strains by bringing beneficial mutations). The ongoing rapid development of next-generation sequencing platforms has provided novel insights into growth kinetics and metabolism of microbes, and thus led to great advances of this technique. In this review, we summarize recent advances in the applications of long-term and short-term ALE techniques mainly for microbial strain engineering, and different modes of ALE are also introduced. Furthermore, we discuss the current limitations of ALE and potential solutions. We believe that the information reviewed here will make a significant contribution to further advancement of ALE.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study.

BACKGROUND: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. RESULTS: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. CONCLUSION: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.