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.

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.

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 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.

[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.

Metabolic engineering of microbes for monoterpenoid production.

Monoterpenoids are an important class of natural products that are derived from the condensation of two five­carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.

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.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

High-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowia lipolytica.

The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non-economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, ß-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the ß-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

[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.

Next-generation metabolic engineering of non-conventional microbial cell factories for carboxylic acid platform chemicals.

Carboxylic acids contain carboxyl groups that can undergo a wide range of chemical transformation. Therefore, they serve as key platform chemicals for the production of high value-added industrial products. Currently, the majority of carboxylic acid platform chemicals is produced predominantly through traditional chemical synthesis. However, these chemical conversion processes are heavily dependent on fossil fuels and often lead to serious environmental pollution. Recently, the rapid development in metabolic engineering of microbes provide a new and promising alternative route for producing carboxylic acids as platform chemicals. We envision that these bio-based manufacturing processes using microbial cell factories will help move the industrial production of carboxylic acid platform chemicals towards a more sustainable, environmentally friendly and economically competitive direction. While Escherichia coli and Saccharomyces cerevisiae have been the workhorses for biochemical production through metabolic engineering, non-conventional microbes are emerging as suitable hosts for producing carboxylic acids to meet the needs of the industries. Here, we review the employment of metabolic engineering strategies on non-conventional microbes to serve as microbial cell factories for the production of industrially important carboxylic acid platform chemicals.

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.

Sustainable production of FAEE biodiesel using the oleaginous yeast Yarrowia lipolytica.

Fatty acid ethyl esters (FAEEs) are fatty acid-derived molecules and serve as an important form of biodiesel. The oleaginous yeast Yarrowia lipolytica is considered an ideal host platform for the production of fatty acid-derived products due to its excellent lipid accumulation capacity. In this proof-of-principle study, several metabolic engineering strategies were applied for the overproduction of FAEE biodiesel in Y. lipolytica. Here, chromosome-based co-overexpression of two heterologous genes, namely, PDC1 (encoding pyruvate decarboxylase) and ADH1 (encoding alcohol dehydrogenase) from Saccharomyces cerevisiae, and the endogenous GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) gene of Y. lipolytica resulted in successful biosynthesis of ethanol at 70.8 mg/L in Y. lipolytica. The engineered Y. lipolytica strain expressing the ethanol synthetic pathway together with a heterologous wax ester synthase (MhWS) exhibited the highest FAEE titer of 360.8 mg/L, which is 3.8-fold higher than that of the control strain when 2% exogenous ethanol was added to the culture medium of Y. lipolytica. Furthermore, a synthetic microbial consortium comprising an engineered Y. lipolytica strain that heterologously expressed MhWS and a S. cerevisiae strain that could provide ethanol as a substrate for the production of the final product in the final engineered Y. lipolytica strain was created in this study. Finally, this synthetic consortium produced FAEE biodiesel at a titer of 4.8 mg/L under the optimum coculture conditions.

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil.

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y. lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil.

BACKGROUND: Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. RESULTS: In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. CONCLUSIONS: This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

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.

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.

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.

[Advances in metabolic engineering for the microbial production of naturally occurring terpenes-limonene and bisabolene: a mini review].

Limonene (C10H16) and bisabolene (C15H24) are both naturally occurring terpenes in plants. Depending on the number of C5 units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.

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.