Reassessing acetyl-CoA supply and NADPH availability for mevalonate biosynthesis from glycerol in Escherichia coli.

Mevalonate is an important platform compound for the biosynthesis of isoprenoids. It can be synthesized from acetyl-CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) by the introduced mvaES operon in Escherichia coli. The influences of E. coli hosts, acetyl-CoA supply, and NADPH availability were assessed and engineered to improve the production titer and yield of mevalonate from glycerol. As a result, E. coli DH5α was found to be the best host with high specific capability and titer of mevalonate from glycerol. Through the engineering of phosphoketolase-phosphotransacetylase (xPK-PTA) bypass and NADPH availability, a final titer of 7.21 g/L with a specific capability of 1.36 g/g dry cell weight was gained in flask culture. Our work could offer new information to metabolically engineer the mevalonate pathway for the efficient production of isoprenoids.

Regulatory molecule cAMP changes cell fitness of the engineered Escherichia coli for terpenoids production.

Terpenoids are a class of natural compounds with many important functions and applications. They are synthesized from a long synthetic pathway of isoprenyl unit coupling with the myriads of terpene synthases. Owing to the catalytic divergence of terpenoids synthesis, microbial production of terpenoids is compromised to the complexity of pathway engineering and suffers from the metabolic engineering burden. In this work, the adaptive Escherichia coli HP variant exhibited a general cell fitness in terpenoid synthesis. Especially, it could yield taxadiene of 193.2 mg/L in a test tube culture, which is a five-fold increase over the production in the wild type E. coli DH5α. Mutational analyses indicated that IS10 insertion in adenylate cyclase CyaA (CyaAHP) resulted in lowering intracellular cyclic AMP (cAMP), which could regulate its receptor protein CRP to rewire cell metabolism and contributed to the improved cell fitness. Our results suggested a way to manipulate cell fitness for terpenoids production and other products.

Improved production of ß-glucan by a T-DNA-based mutant of Aureobasidium pullulans.

To improve ß-1,3-1,6-D-glucan (ß-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and ß-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and ß-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored ß-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved ß-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • ß-Glucan produced by the mutant was significantly improved.

Extension of cell membrane boosting squalene production in the engineered Escherichia coli.

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.

Triton X-100 improves co-production of ß-1,3-D-glucan and pullulan by Aureobasidium pullulans.

The effects of several surfactants on the biosynthesis of ß-1,3-D-glucan (ß-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase ß-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased ß-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in ß-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of ß-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of ß-glucan and pullulan in the presence of 5 g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on ß-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased ß-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in ß-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Challenges and tackles in metabolic engineering for microbial production of carotenoids.

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.

Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan.

The effect of sodium chloride (NaCl) on pullulan production by batch culture of Aureobasidium pullulans CCTCC M 2012259 was investigated. NaCl at 3 g/L improved the pullulan titer by 26.7% but reduced the molecular weight of pullulan to only 46.8% of that obtained in the control without NaCl. In order to elucidate the physiological mechanism underlying the effect of NaCl on pullulan production, assays of key enzyme activity, gene expression, energy metabolism, and intracellular uridine diphosphate glucose (UDP-glucose) content were performed. Results indicated that NaCl increased the activities of α-phosphoglucose mutase and glucosyltransferase involved in pullulan biosynthesis, increased the activities of α-amylase being responsible for pullulan degradation, upregulated the transcriptional levels of pgm1, fks, and amy2 genes, enhanced the driving force for ATP supply, and helped to maintain intracellular UDP-glucose at a high level in A. pullulans CCTCC M 2012259. All these results illuminate the reason by which NaCl increases pullulan titer but reduces the molecular weight of pullulan.

Improved S-adenosylmethionine and glutathione biosynthesis by heterologous expression of an ATP6 gene in Candida utilis.

ATP is indispensable to the biosynthesis of both S-adenosylmethionine (SAM) and glutathione (GSH) in yeast cells. To improve ATP supply for overproduction of SAM and GSH in Candida utilis CCTCC M 209298, an exogenous ATP6 gene from Arabidopsis thaliana was expressed in the parental strain to construct the mutant C. utilis ATP6 by genomic integration. The maximal production of SAM and GSH in the mutant increased by 46.6 and 28.7%, respectively, when compared with those obtained in the parental strain. The mechanism underlying improved SAM and GSH biosynthesis by exogenous ATP6 gene expression revealed that the mutant had higher activities of key enzymes involved in SAM and GSH biosynthesis as well as energy metabolism. Increased NADH availability and F0 F1 -ATPase activity subsequently resulted in improved ATP regeneration and intracellular ATP supply for SAM and GSH overproduction. The present study not only developed an effective method for improving SAM and GSH biosynthesis by energy metabolism regulation, but also offered a novel approach for efficient production of similar energy-consuming products in eukaryotic cells.

The mechanism of improved intracellular organic selenium and glutathione contents in selenium-enriched Candida utilis by acid stress.

Batch culture of Candida utilis CCTCC M 209298 for the preparation of selenium (Se)-enriched yeast was carried out under different pH conditions, and maximal intracellular organic Se and glutathione (GSH) contents were obtained in a moderate acid stress environment (pH 3.5). In order to elucidate the physiological mechanism of improved performance of Se-enriched yeast by acid stress, assays of the key enzymes involved in GSH biosynthesis and determinations of energy supply and regeneration were performed. The results indicated that moderate acid stress increased the activity of γ-glutamylcysteine synthetase and the ratios of NADH/NAD+ and ATP/ADP, although no significant changes in intracellular pH were observed. In addition, the molecular mechanism of moderate acid stress favoring the improvement of Se-yeast performance was revealed by comparing whole transcriptomes of yeast cells cultured at pH 3.5 and 5.5. Comparative analysis of RNA-Seq data indicated that 882 genes were significantly up-regulated by moderate acid stress. Functional annotation of the up-regulated genes based on gene ontology and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway showed that these genes are involved in ATP synthesis and sulfur metabolism, including the biosynthesis of methionine, cysteine, and GSH in yeast cells. Increased intracellular ATP supply and more amounts of sulfur-containing substances in turn contributed to Na2SeO3 assimilation and biotransformation, which ultimately improved the performance of the Se-enriched C. utilis.

Efficient pullulan production by bioconversion using Aureobasidium pullulans as the whole-cell catalyst.

In this study, pullulan production was achieved by whole-cell bioconversion with Aureobasidium pullulans CCTCC M 2012259. Response surface methodology was applied to optimize the seed medium for incubating cells with high capability of pullulan bioconversion. Three medium components, namely, yeast extract, MgSO4·7H2O, and glucose were identified by Plackett-Berman design as significant factors affecting the cells' pullulan bioconversion capability. A three-level Box-Behnken design was then employed to determine the optimal levels of the three components. A mathematical model was developed to show the influence of each medium component and its effects on the cells' pullulan bioconversion capability. The model predicted a maximum pullulan bioconversion capability of 32.28 mg/g/h at the optimal yeast extract, MgSO4·7H2O, and glucose concentrations of 3.57, 0.18, and 63.97 g/l, respectively. The validation experiments showed that the cells' pullulan bioconversion capability was improved by 23.1% when the optimal medium was used, as compared with that obtained with the basic medium. Subsequently, the gene expression and activities of the key enzymes involved in pullulan biosynthesis were evaluated. When the optimal medium was employed, the transcriptional levels of pgm1 and fks were up-regulated by 2.5- and 1.2-fold, respectively, and the α-phosphoglucose mutase and glucosyltransferase activities were increased by 17 and 19%, respectively, when compared with those achieved using the basic medium. These results indicated that pullulan bioconversion using A. pullulans CCTCC M 2012259 as the whole-cell catalyst is an attractive approach for efficient pullulan production and can be applied for the production of other polysaccharides.

Glutathione is involved in physiological response of Candida utilis to acid stress.

Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH 3.5 for 6 h) and a short-term severe acid challenge (pH 1.5 for 2 h) were conducted at 18 h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms.

Whole-crop biorefinery of corn biomass for pullulan production by Aureobasidium pullulans.

In the present study, corn starch, cob, and straw were biorefined and used as feedstocks for the production of pullulan. The titer and molecular weight (Mw) of pullulan significantly decreased when corn cob and straw hydrolysates were utilized by the parental strain Aureobasidium pullulans CCTCC M 2012259 (PS). Based on adaptive laboratory evolution of PS, an evolved strain A. pullulans EV6 with strong adaptability to the whole corn biomass hydrolysate and high capability of pullulan biosynthesis was screened. Batch pullulan fermentation results indicated that EV6 produced an increased titer of pullulan with a higher Mw than PS. The underlying reasons for these increases were revealed by assaying key enzymes activities and measuring intracellular uridine diphosphate glucose levels. Subsequently, whole-crop biorefinery of corn biomass was conducted, and the results confirmed that whole corn crop has immense potential for efficient pullulan production.

Screening of Selenium/Glutathione-Enriched Candida utilis and Its Anti-inflammatory and Antioxidant Activities in Mice.

This study aimed to screen a mutant of Candida utilis SE-172 with high selenite tolerance and glutathione (GSH) biosynthesis capability via 60Co γ-radiation mutagenesis to prepare selenium (Se)-enriched yeast. The maximal intracellular contents of GSH and organic Se of 22.94 mg/g and 1308.1 µg/g were obtained, respectively, under a batch culture of SE-172. The physiological mechanism underlying increased GSH and organic Se contents in Se/GSH-enriched C. utilis SE-172 was revealed based on assaying activities of γ-glutamylcysteine synthase (γ-GCS) involved in GSH biosynthesis and selenophosphate synthase (SPS) related to organic Se bioconversion, and by determining intracellular ATP and NADH contents and ATP/ADP and NADH/NAD+ ratios associated with energy supply and regeneration. Moreover, the effect of this selenized yeast on anti-inflammatory and antioxidant activities in mice with colitis was investigated. The supplementation of Se/GSH-enriched yeast decreased the dextran sodium sulfate-induced damage to colon tissues, reduced the expression of pro-inflammatory factors [interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α)] in serum, increased the antioxidant-related enzyme [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)] activities, and decreased the malondialdehyde content in colon. The Se/GSH-enriched C. utilis SE-172 showed potent anti-inflammatory and antioxidant activities in mice with colitis.

MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans.

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for ß-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

Efficient pullulan production by Aureobasidium pullulans using cost-effective substrates.

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

[Construction and optimization of squalene epoxide synthetic pathway in Escherichia coli].

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.

Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil.

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., Escherichia coli) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in E. coli, and the best engineered E. coli NA-IS3D strain could produce α-santalene at a titer of 412 mg·L-1. Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indole-associated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of tnaA could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg·L-1 in E. coli tnaA- NA-IS3D. Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.

Metabolic flux and transcriptome analyses provide insights into the mechanism underlying zinc sulfate improved ß-1,3-D-glucan production by Aureobasidium pullulans.

The effects of zinc sulfate at various concentrations on ß-1,3-D-glucan (ß-glucan) and pullulan production were investigated in flasks, and 0.1 g/L zinc sulfate was found to be the optimum concentration favoring increased ß-glucan production. When batch culture of Aureobasidium pullulans CCTCC M 2012259 with 0.1 g/L zinc sulfate was carried out, the maximum dry biomass decreased by 16.9% while ß-glucan production significantly increased by 120.5%, compared to results obtained from the control without zinc sulfate addition. To reveal the mechanism underlying zinc sulfate improved ß-glucan production, both metabolic flux analysis and RNA-seq analysis were performed. The results indicated that zinc sulfate decreased carbon flux towards biomass formation and ATP supply, down-regulated genes associated with membrane part and cellular components organization, leading to a decrease in dry cell weight. However, zinc sulfate increased metabolic flux towards ß-glucan biosynthesis, up-regulated genes related to glycan biosynthesis and nucleotide metabolism, resulting in improved ß-glucan production. This study provides insights into the changes in the metabolism of A. pullulans in response to zinc sulfate, and can serve as a valuable reference of genetic information for improving the production of polysaccharides through metabolic engineering.

Transcriptome analysis reveals the mechanism underlying improved glutathione biosynthesis and secretion in Candida utilis during selenium enrichment.

The effect of sodium selenite on batch culture of Candida utilis CCTCC M 209298 was investigated. Cell growth was inhibited while glutathione biosynthesis and secretion were improved during selenium enrichment. To reveal the mechanism underlying the decrease in biomass and the increase in glutathione, both metabolic flux analysis of key intermediates involved in glutathione metabolic pathway and transcriptome analysis of C. utilis by RNA-seq were carried out for selenized cells and the control without selenium enrichment. Results indicated that sodium selenite decreased carbon fluxes towards biomass but increased fluxes towards amino acids for the biosynthesis of glutathione and related amino acids. Selenium enrichment down-regulated a large number of genes involved in cell components and the cell cycle, resulting in decreased biomass as well as increased cell permeability. Moreover, several genes associated with transportation, binding, and mitochondrial and ribosomal functions for energy metabolism and protein synthesis were up-regulated in the presence of sodium selenite. All of these results disclosed the physiological response of C. utilis to sodium selenite.

Disruption of por1 gene in Candida utilis improves co-production of S-adenosylmethionine and glutathione.

The por1 gene encoding one of the mitochondrial porin channels in C. utilis CCTCC M 209298 was disrupted using a homologous recombination method. The co-production of S-adenosylmethionine (SAM) and glutathione (GSH) in the mutant C. utilis Δpor1 increased by 34.9% and 25.1%, respectively, during batch and fed-batch fermentation, relative to the parental strain. The average oxygen consumption rate, activities of key enzymes involved in SAM and GSH biosynthesis, levels of intracellular cofactors such as NADH and ATP, and carbon fluxes of key metabolites were compared between the parental strain and the Δpor1 mutant. The disruption of por1 gene increased the rate of mitochondrial respiration, increased the activities of both methionine adenosyltransferase and γ-glutamylcysteine synthetase, and enhanced the supply of energy and substrates for SAM and GSH biosynthesis, all of which favored the overproduction of SAM and GSH in the Δpor1 mutant.