Synthetic redesign of Escherichia coli W for faster metabolism of sugarcane molasses.

BACKGROUND: Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. RESULTS: In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. CONCLUSIONS: Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.

System analysis of Lipomyces starkeyi during growth on various plant-based sugars.

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels. KEY POINTS: • L. starkeyi metabolism reprograms for consumption of different plant-based sugars. • Non-specific regulation was observed during growth on cellobiose. • L. starkeyi secretes ß-glucosidases for extracellular hydrolysis of cellobiose.

Sugar uptake, metabolism, and chloride secretion in the rectal gland of the spiny dogfish Squalus acanthias.

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.

Model-assisted comparison of sugar accumulation patterns in ten fleshy fruits highlights differences between herbaceous and woody species.

BACKGROUND AND AIMS: Sugar concentration is a key determinant of fruit quality. Soluble sugars and starch concentrations in fruits vary greatly from one species to another. The aim of this study was to investigate similarities and differences in sugar accumulation strategies across ten contrasting fruit species using a modelling approach. METHODS: We developed a coarse-grained model of primary metabolism based on the description of the main metabolic and hydraulic processes (synthesis of compounds other than sugar and starch, synthesis and hydrolysis of starch, and water dilution) involved in the accumulation of soluble sugars during fruit development. KEY RESULTS: Statistical analyses based on metabolic rates separated the species into six groups according to the rate of synthesis of compounds other than sugar and starch. Herbaceous species (cucumber, tomato, eggplant, pepper and strawberry) were characterized by a higher synthesis rate than woody species (apple, nectarine, clementine, grape and kiwifruit). Inspection of the dynamics of the processes involved in sugar accumulation revealed that net sugar importation, metabolism and dilution processes were remarkably synchronous in most herbaceous plants, whereas in kiwifruit, apple and nectarine, processes related to starch metabolism were temporally separated from other processes. Strawberry, clementine and grape showed a distinct dynamic compared with all other species. CONCLUSIONS: Overall, these results provide fresh insights into species-specific regulatory strategies and into the role of starch metabolism in the accumulation of soluble sugars in fleshy fruits. In particular, inter-specific differences in development period shape the co-ordination of metabolic processes and affect priorities for carbon allocation across species. The six metabolic groups identified by our analysis do not show a clear separation into climacteric and non-climacteric species, possibly suggesting that the metabolic processes related to sugar concentration are not greatly affected by ethylene-associated events.

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Plastidial Folate Prevents Starch Biosynthesis Triggered by Sugar Influx into Non-Photosynthetic Plastids of Arabidopsis.

Regulation of sucrose-starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism.

Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth.

Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.g., W29) are known to be slow-growing in fructose while others (e.g., H222) grow rapidly under the same conditions. Here, we retrieved 24 proteins of the Sugar Porter family from these two strains, and determined that at least six of these proteins can function as hexose transporters in the heterologous host Saccharomyces cerevisiae EBY.VW4000. Transcriptional studies and deletion analysis in Y. lipolytica indicated that two genes, YHT1 and YHT4, are probably the main players in both strains, with a similar role in the uptake of glucose, fructose, and mannose at various concentrations. The other four genes appear to constitute a set of 'reservoir' hexose transporters with an as-yet unclear physiological role. Furthermore, through examining Sugar Porters of the entire Yarrowia clade, we show that they constitute a dynamic family, within which hexose transport genes have been duplicated and lost several times. Our phylogenetic analyses support the existence of at least three distinct evolutionary groups of transporters which allow yeasts to grow on hexoses. In addition to the well-known and widespread Hxt-type transporters (which are not essential in Y. lipolytica), we highlight a second group of transporters, represented by Yht1, which are phylogenetically related to sensors that play a regulatory role in S. cerevisiae, and a third group, represented by Yht4, previously thought to contain only high-affinity glucose transporters related to Hgt1of Kluyveromyces lactis.

Transporter engineering in biomass utilization by yeast.

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.

Sugar uptake by the solventogenic clostridia.

The acetone-butanol-ethanol fermentation of solventogenic clostridia was operated as a successful, worldwide industrial process during the first half of the twentieth century, but went into decline for economic reasons. The recent resurgence in interest in the fermentation has been due principally to the recognised potential of butanol as a biofuel, and development of reliable molecular tools has encouraged realistic prospects of bacterial strains being engineered to optimise fermentation performance. In order to minimise costs, emphasis is being placed on waste feedstock streams containing a range of fermentable carbohydrates. It is therefore important to develop a detailed understanding of the mechanisms of carbohydrate uptake so that effective engineering strategies can be identified. This review surveys present knowledge of sugar uptake and its control in solventogenic clostridia. The major mechanism of sugar uptake is the PEP-dependent phosphotransferase system (PTS), which both transports and phosphorylates its sugar substrates and plays a central role in metabolic regulation. Clostridial genome sequences have indicated the presence of numerous phosphotransferase systems for uptake of hexose sugars, hexose derivatives and disaccharides. On the other hand, uptake of sugars such as pentoses occurs via non-PTS mechanisms. Progress in characterization of clostridial sugar transporters and manipulation of control mechanisms to optimise sugar fermentation is described.

Structural and biochemical insights of xylose MFS and SWEET transporters in microbial cell factories: challenges to lignocellulosic hydrolysates fermentation.

The production of bioethanol from lignocellulosic biomass requires the efficient conversion of glucose and xylose to ethanol, a process that depends on the ability of microorganisms to internalize these sugars. Although glucose transporters exist in several species, xylose transporters are less common. Several types of transporters have been identified in diverse microorganisms, including members of the Major Facilitator Superfamily (MFS) and Sugars Will Eventually be Exported Transporter (SWEET) families. Considering that Saccharomyces cerevisiae lacks an effective xylose transport system, engineered yeast strains capable of efficiently consuming this sugar are critical for obtaining high ethanol yields. This article reviews the structure-function relationship of sugar transporters from the MFS and SWEET families. It provides information on several tools and approaches used to identify and characterize them to optimize xylose consumption and, consequently, second-generation ethanol production.

A Prunus avium L. Infusion Inhibits Sugar Uptake and Counteracts Oxidative Stress-Induced Stimulation of Glucose Uptake by Intestinal Epithelial (Caco-2) Cells.

Sweet cherry (Prunus avium L.) is among the most valued fruits due to its organoleptic properties and nutritional worth. Cherry stems are rich in bioactive compounds, known for their anti-inflammatory and antioxidant properties. Innumerable studies have indicated that some bioactive compounds can modulate sugar absorption in the small intestine. In this study, the phenolic profile of a cherry stem infusion was investigated, as well as its capacity to modulate intestinal glucose and fructose transport in Caco-2 cells. Long-term (24 h) exposure to cherry stem infusion (25%, v/v) significantly reduced glucose (3H-DG) and fructose (14C-FRU) apical uptake, reduced the apical-to-basolateral Papp to 3H-DG, and decreased mRNA expression levels of the sugar transporters SGLT1, GLUT2 and GLUT5. Oxidative stress (induced by tert-butyl hydroperoxide) caused an increase in 3H-DG uptake, which was abolished by the cherry stem infusion. These findings suggest that cherry stem infusion can reduce the intestinal absorption of both glucose and fructose by decreasing the gene expression of their membrane transporters. Moreover, this infusion also appears to be able to counteract the stimulatory effect of oxidative stress upon glucose intestinal uptake. Therefore, it can be a potentially useful compound for controlling hyperglycemia, especially in the presence of increased intestinal oxidative stress levels.

Pulsed Electric Field and Freeze-Thawing Pretreatments for Sugar Uptake Modulation during Osmotic Dehydration of Mango.

Osmotic dehydration kinetics depends on food tissue microstructure; thus, modulation of mango porosity could help selectively enhance water removal over sugar gain. In this present study, pretreatments of freeze-thawing (freezing at -36 °C for 2 weeks and thawing at 4 °C for 24 h) and pulsed electric field (1 kV/cm, 10 and 30 pulse numbers), were applied to mango 1 cm-thickness slices prior to osmotic dehydration conducted at 40 °C for 4 h. Three different 60 °Brix agave syrup solutions with or without added polysaccharides (inulin or xanthan gum) were used in the osmotic dehydration operation. Water loss (WL), sugar gain (SG) and microstructure images were used to compare the effects of pretreatments on mango osmotic dehydration efficiency. Results indicated that pulsed electric field (PEF) pretreatment increased slightly WL during osmotic dehydration, contrary to freeze-thawing (F-T), which for most cases led to a decrease. As for solids uptake, due to higher damage induced by F-T to mango tissue, SG was higher than for fresh and PEF pretreated mangoes. Using xanthan gum as additive to agave syrup solution, helped to decrease sugar uptake in frozen-thawed mango due to an increase in solution viscosity. A similar WL/SG ratio was obtained with frozen-thawed mango in solution with xanthan gum. Therefore, in the case of frozen-thawed mango, it is recommended to use an osmotic solution with high viscosity to obtain low sugar uptake in the final product. The novelty of this contribution is twofold: (i) using pretreatments (F-T or PEF) to minimize sugar uptake during osmotic dehydration, and (ii) using agave syrup with added polysaccharides to enrich final product with inulin.

Beer fermentation performance and sugar uptake of Saccharomycopsis fibuligera-A novel option for low-alcohol beer.

There is a growing trend for beers with novel flavor profiles, as consumers demand a more diversified product range. Such beers can be produced by using non-Saccharomyces yeasts. The yeast species Saccharomycopsis fibuligera is known to produce exceptionally pleasant plum and berry flavors during brewer's wort fermentation while its mycelia growth is most likely a technological challenge in industrial-scale brewing. To better understand and optimize the physiological properties of this yeast species during the brewing process, maltose and maltotriose uptake activity trials were performed. These revealed the existence of active transmembrane transporters for maltose in addition to the known extracellular amylase system. Furthermore, a single cell isolate of S. fibuligera was cultured, which showed significantly less mycelial growth during propagation and fermentation compared to the mother culture and would therefore be much more suitable for application on an industrial scale due to its better flocculation and clarification properties. Genetic differences between the two cultures could not be detected in a (GTG)5 rep-PCR fingerprint and there was hardly any difference in the fermentation process, sugar utilization and flavor profiles of the beers. Accordingly, the characteristic plum and berry flavor could also be perceived by using the culture from the single cell isolate, which was complemented by a dried fruit flavor. A fermentation temperature of 20°C at an original gravity of 10 °P proved to be optimal for producing a low-alcohol beer at around 0.8% (v/v) by applying the S. fibuligera yeast culture from the single cell isolate.

Green/Roasted Coffee and Silverskin Extracts Inhibit Sugar Absorption by Human Intestinal Epithelial (Caco-2) Cells by Decreasing GLUT2 Gene Expression.

Moderate coffee ingestion has been associated with a decrease in type 2 diabetes risk, mainly due to its richness in chlorogenic acids (CGA). To explore this, extracts of green beans, roasted beans, and silverskin were prepared by aqueous ultrasound-assisted extraction and characterized by a reversed-phase high-performance liquid chromatography-photodiode array detector (RP-HPLC-DAD). The effects on the uptake of glucose and fructose by human intestinal epithelial (Caco-2) cells and the influence on the expression of sugar transporter genes (by RT-qPCR) were investigated and compared. The uptake of 3H-deoxy-D-glucose and 14C-fructose by Caco-2 cells was significantly reduced by all the extracts, with green coffee (which also contained higher amounts of CGA) achieving the highest efficiency. Although silverskin presented the lowest amounts of CGA and caffeine, it promoted an inhibitory effect similar to the effects of green/roasted beans. In the case of glucose uptake, the effect was even higher than for roasted coffee. This activity is explained by the ability of the extracts to markedly decrease GLUT2, but not GLUT5 gene expression. In addition, a decrease in SGLT1 gene expression was also found for all extracts, although not at a statistically significant rate for silverskin. This study also revealed a synergistic inhibitory effect of caffeine and 5-CQA on the uptake of sugars. Thus, silverskin appears as an interesting alternative to coffee, since the valorization of this by-product also contributes to the sustainability of the coffee chain.

In vivo Inhibition of the 3-Dehydroquinate Synthase by 7-Deoxysedoheptulose Depends on Promiscuous Uptake by Sugar Transporters in Cyanobacteria.

7-Deoxysedoheptulose (7dSh) is a bioactive deoxy-sugar actively excreted by the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) but also Streptomyces setonensis. In our previous publications we have shown that in S. elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved. Additionally, we showed that 7dSh inhibits the growth of cyanobacteria, but also the growth of plants and fungi, presumably by inhibiting the 3-dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, as the substrate of this enzyme strongly accumulates in cells treated with 7dSh. In this study, by using purified DHQS of Anabaena variabilis ATCC 29413 (A. variabilis) we biochemically confirmed that 7dSh is a competitive inhibitor of this enzyme. By analyzing the effect of 7dSh on a subset of cyanobacteria from all the five subsections, we identified different species whose growth was inhibited by 7dSh. We also found that in some of the susceptible cyanobacteria import of 7dSh is mediated by structurally different and promiscuous transporters: 7dSh can be taken up by the fructose ABC-transporter in A. variabilis and via the glucose permease in Synechocystis sp. PCC 6803 (Synechocystis sp.). In both cases, an effective uptake and thereby intracellular enrichment of 7dSh was essential for the inhibitory activity. Importantly, spontaneous mutations in the sugar transporters of A. variabilis and Synechocystis sp. not only disabled growth of the two strains on fructose and glucose, respectively, but also almost abolished their sensitivity to 7dSh. Although we have clearly shown in these examples that the effective uptake plays an essential role in the inhibitory effect of 7dSh, questions remain about how 7dSh resistance works in other (cyano)bacteria. Also, the involvement of a putative ribokinase in 7dSh resistance in the producer strain S. elongatus remained to be further investigated. Overall, these data establish 7dSh as the first allelochemical targeting the shikimate pathway in other cyanobacteria and plants and suggest a role of 7dSh in niche competition.

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose.

BACKGROUND: Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry. RESULTS: We selected Myceliophthora thermophila, a cellulolytic thermophilic filamentous fungus for metabolic engineering to produce ethanol from glucose and cellobiose. Ethanol production was increased by 57% from glucose but not cellobiose after introduction of ScADH1 into the wild-type (WT) strain. Further overexpression of a glucose transporter GLT-1 or the cellodextrin transport system (CDT-1/CDT-2) from N. crassa increased ethanol production by 131% from glucose or by 200% from cellobiose, respectively. Transcriptomic analysis of the engineered cellobiose-utilizing strain and WT when grown on cellobiose showed that genes involved in oxidation-reduction reactions and the stress response were downregulated, whereas those involved in protein biosynthesis were upregulated in this effective ethanol production strain. Turning down the expression of pyc gene results the final engineered strain with the ethanol production was further increased by 23%, reaching up to 11.3 g/L on cellobiose. CONCLUSIONS: This is the first attempt to engineer the cellulolytic fungus M. thermophila to produce bioethanol from biomass-derived sugars such as glucose and cellobiose. The ethanol production can be improved about 4 times up to 11 grams per liter on cellobiose after a couple of genetic engineering. These results show that M. thermophila is a promising platform for bioethanol production from cellulosic materials in the future.

Mining and fine-tuning sugar uptake system for titer improvement of milbemycins in Streptomyces bingchenggensis.

Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase, when antibiotics are extensively produced. Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides, while the low yield and titer impede their broad applications in agricultural field. Considering that inadequate sugar uptake generally hinders titer improvement of desired products, we mined the underlying sugar uptake systems and fine-tuned their expression in this work. First, we screened the candidates at both genomic and transcriptomic level in S. bingchenggensis. Then, two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly. Next, the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5, resulting in a maximal milbemycin A3/A4 titer increase by 36.9% to 3321 mg/L. Finally, TP2 and TP5 were broadly fine-tuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus, leading to a maximal titer improvement of 34.1% and 52.6% for avermectin B1a and nemadectin, respectively. This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.

A Growth-Based Screening System for Hexose Transporters in Yeast.

As the simplest eukaryotic model system, the unicellular yeast Saccharomyces cerevisiae is ideally suited for quick and simple functional studies as well as for high-throughput screening. We generated a strain deficient for all endogenous hexose transporters, which has been successfully used to clone, characterize, and engineer carbohydrate transporters from different source organisms. Here we present basic protocols for handling this strain and characterizing sugar transporters heterologously expressed in it.

Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum.

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.

Cardiotrophin-1 decreases intestinal sugar uptake in mice and in Caco-2 cells.

AIM: Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines with a key role in glucose and lipid metabolism. In the current investigation, we examined the in vivo and in vitro effects of CT-1 treatment on intestinal sugar absorption in different experimental models. METHODS: rCT-1 effects on α-Methyl-D-glucoside uptake were assessed in everted intestinal rings from wild-type and CT-1(-/-) mice and in Caco-2 cells. rCT-1 actions on SGLT-1 expression in brush border membrane vesicles and the identification of the potential signalling pathways involved were determined by Western blot. RESULTS: In vivo administration (0.2 mg kg(-1) ) of rCT-1 caused a significant decrease on α-Methyl-D-glucoside uptake in everted intestinal rings from wild-type and CT-1(-/-) mice after short-term and long-term treatments. Similarly, in vitro treatment (1-50 ng mL(-1) ) with rCT-1 reduced α-Methyl-D-glucoside uptake in everted intestinal rings. In Caco-2 cells, rCT-1 treatment (20 ng mL(-1) , 1 and 24 h) lowered apical uptake of α-Methyl-D-glucoside in parallel with a decrease on SGLT-1 protein expression. rCT-1 promoted the phosphorylation of STAT-3 after 5 and 15 min treatment, but inhibited the activation by phosphorylation of AMPK after 30 and 60 min. Interestingly, pre-treatment with the JAK/STAT inhibitor (AG490) and with the AMPK activator (AICAR) reversed the inhibitory effects of rCT-1 on α-Methyl-D-glucoside uptake. AICAR also prevented the inhibition of SGLT-1 observed in rCT-1-treated cells. CONCLUSIONS: CT-1 inhibits intestinal sugar absorption by the reduction of SGLT-1 levels through the AMPK pathway, which could also contribute to explain the hypoglycaemic and anti-obesity properties of CT-1.