Pdc Genes Research Articles

Study on the mechanism on synthesis of higher alcohols in Wickerhamomyces anomalus under ethanol stress

AbstractHigher alcohols (phenylethyl alcohol and isoamyl alcohol), as the crucial aroma compounds, have been found to associate with the sensory properties of various alcoholic products. The Ehrlich and Harris pathways are the main pathways for yeast to produce phenylethyl alcohol and isoamyl alcohol during the brewing process of alcoholic products, whereas the specific changes of the Ehrlich and Harris pathways in Wickerhamomyces anomalus under ethanol stress are still unclear. In this study, W. anomalus NCU003 with a high capacity for aroma compound production was treated with ethanol stress at different concentrations (3%, v/v, 6%, v/v and 9%, v/v) to study the effects on the expression levels of genes and metabolite content in the Ehrlich and Harris pathways. The results found that ethanol stress inhibited the production of phenylethyl alcohol and isoamyl alcohol, and the content decreased with the increase of ethanol concentrations. In the meantime, the up‐regulation of ARO1, CS, ARO8, Leu1, BAT2 and ilvs family genes in the Ehrlich and Harris pathways could lead to an increase in the synthesis of important precursor substances during the synthesis of phenylethyl alcohol and isoamyl alcohol. However, the significant down‐regulation of ARO10, PDC, ADH1 and AHD2 genes was the main reason for the low production of phenylethyl alcohol and isoamyl alcohol. These results could provide a theoretical reference for the construction of genetic engineering strains of W. anomalus in the future so as to reasonably control the content of higher alcohols during the brewing process of alcoholic products.

LOX and AAT Genes Affect the Aroma of “Xiaobai” Apricot during Postharvest Storage

“Xiaobai” apricot is one of the most popular fruits in China. However, during postharvest storage, fruit aroma loss occurs easily. In this study, factors affecting the aroma changes in different ripening stages of “Xiaobai” apricot during postharvest storage were searched. Immature and mature “Xiaobai” apricot samples were collected and monitored for sensory changes during postharvest storage. After 25 days of storage, the aromatic ester and alcohol abundance in mature showed a decrease, related to the loss of characteristic aroma. Immature fruit still tasted sour with an indistinct characteristic aroma, as a result of decreased alcohol and increased acid abundance. The ATT, ADH, PDC, and LOX genes were identified, and their expression levels were detected at different ripening stages during storage. The correlation analysis showed that the expression of LOX and ATT was positively correlated with the variation of aromatic ester ( P < 0.05 ), a key factor affecting the apricot aroma during postharvest storage.

The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate.

Dekkera bruxellensis has been studied for several aspects of its metabolism over the past years, which has expanded our comprehension on its importance to industrial fermentation processes and uncovered its industrial relevance. Acetate is a metabolite often found in D. bruxellensis aerobic cultivations, whereas its production is linked to decreased ethanol yields. In a previous work, we aimed to understand how acetate metabolism affected the fermentation capacity of D. bruxellensis. In the present work, we evaluated the role of acetate metabolism in respiring cells using ammonium or nitrate as nitrogen sources. Our results showed that galactose is a strictly respiratory sugar and that a relevant part of its carbon is lost, while the remaining is metabolised through the Pdh bypass pathway before being assimilated into biomass. When this pathway was blocked, yeast growth was reduced while more carbon was assimilated to the biomass. In nitrate, more acetate was produced as expected, which increased carbon assimilation, although less galactose was uptaken from the medium. This scenario was not affected by the Pdh bypass inhibition. The confirmation that acetate production was crucial for carbon assimilation was brought by cultivations in pyruvate. All physiological data were connected to the expression patterns of PFK1, PDC1, ADH1, ALD3, ALD5 and ATP1 genes. Other respiring carbon sources could only be properly used by the cells when some external acetate was supplied. Therefore, the results reported herein helped in providing valuable contributions to the understanding of the oxidative metabolism in this potential industrial yeast.

Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate.

Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2g/L lactate and 16.9 ± 0.3g/L ethanol using 48g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6g/L lactate and 12.9 ± 0.8g/L ethanol as well as 36.2 ± 1.0g/L lactate and 40.3 ± 0.3g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0g/L/h and 2.2 ± 0.0g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1g/L D-lactate and 27.7 ± 0.2g/L ethanol as well as 42.8 ± 0.0g/L D-lactate and 53.1 ± 0.7g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.

Metabolic Engineering of Zymomonas mobilis for Acetoin Production by Carbon Redistribution and Cofactor Balance

Biorefinery to produce value-added biochemicals offers a promising alternative to meet our sustainable energy and environmental goals. Acetoin is widely used in the food and cosmetic industries as taste and fragrance enhancer. The generally regarded as safe (GRAS) bacterium Zymomonas mobilis produces acetoin as an extracellular product under aerobic conditions. In this study, metabolic engineering strategies were applied including redistributing the carbon flux to acetoin and manipulating the NADH levels. To improve the acetoin level, a heterologous acetoin pathway was first introduced into Z. mobilis, which contained genes encoding acetolactate synthase (Als) and acetolactate decarboxylase (AldC) driven by a strong native promoter Pgap. Then a gene encoding water-forming NADH oxidase (NoxE) was introduced for NADH cofactor balance. The recombinant Z. mobilis strain containing both an artificial acetoin operon and the noxE greatly enhanced acetoin production with maximum titer reaching 8.8 g/L and the productivity of 0.34 g∙L−1∙h−1. In addition, the strategies to delete ndh gene for redox balance by native I-F CRISPR-Cas system and to redirect carbon from ethanol production to acetoin biosynthesis through a dcas12a-based CRISPRi system targeting pdc gene laid a foundation to help construct an acetoin producer in the future. This study thus provides an informative strategy and method to harness the NADH levels for biorefinery and synthetic biology studies in Z. mobilis.

Using transcriptomics to reveal the molecular mechanism of higher alcohol metabolism in Saccharomyces cerevisiae

High alcohols are important flavor compounds in alcoholic beverages, but high concentration of high alcohols is harmful to human health. Higher alcohol synthesis pathways in brewing microorganisms have been investigated, but the interactions between key genes remain to be explored, especially in industrial strains of Saccharomyces cerevisiae. The PDC1 gene was considered to be the main encoding gene of α-keto acid decarboxylase, and its deletion resulted in a 92.23% increase in isobutanol production and a 14.89% decrease in isoamyl alcohol production. Transcriptome sequencing was used to explore the effects of PDC1 gene deletion on global gene transcription levels, and deletion strategies were utilized to verify the effects of differential genes on higher alcohol production. Deletion of differential gene HMLALPHA2 increased isobutanol production by 26.23%, and decreased isoamyl alcohol and 2-phenylethanol production by 30.31% and 22.35%, respectively. The THI2, THI4, THI20 genes were proved to be related to n-propanol synthesis. In addition, HMRA2 and SIR3 genes were found to influence isoamyl alcohol synthesis pathways, and their deletion increased isoamyl alcohol production by 25.18% and 21.76%. Our discovery of new target genes is useful for elucidating the molecular mechanisms of higher alcohols and the construction of novel low-producing higher alcohol strains.

Transcriptomic profiling analysis to identify genes associated with PA biosynthesis and insolubilization in the late stage of fruit development in C-PCNA persimmon

PA-enhanced content causes astringency in persimmon fruit. PCNA persimmons can lose their astringency naturally and they become edible when still on the tree, which allows for conserves of physical and financial resources. C-PCNA persimmon originates in China. Its deastringency trait primarily depends on decreased PA biosynthesis and PA insolubilization at the late stage of fruit development. Although some genes and transcription factors that may be involved in the deastringency of C-PCNA persimmon have been reported, the expression patterns of these genes during the key deastringency stage are reported less. To investigate the variation in PA contents and the expression patterns of deastringency-related genes during typical C-PCNA persimmon ‘Xiaoguo-tianshi’ fruit development and ripening, PA content and transcriptional profiling were carried out at five late stages from 70 to 160 DAF. The combinational analysis phenotype, PA content, and DEG enrichment revealed that 120–140 DAF and 140–160 DAF were the critical phases for PA biosynthesis reduction and PA insolubilization, respectively. The expression of PA biosynthesis-associated genes indicated that the downregulation of the ANR gene at 140–160 DAF may be associated with PA biosynthesis and is decreased by inhibiting its precursor cis-flavan-3-ols. We also found that a decrease in acetaldehyde metabolism-associated ALDH genes and an increase in ADH and PDC genes might result in C-PCNA persimmon PA insolubilization. In addition, a few MYB-bHLH-WD40 (MBW) homologous transcription factors in persimmon might play important roles in persimmon PA accumulation. Furthermore, combined coexpression network analysis and phylogenetic analysis of MBW suggested that three putative transcription factors WD40 (evm.TU.contig1.155), MYB (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203), might connect and co-regulate both PA biosynthesis and its insolubilization in C-PCNA persimmon. The present study elucidated transcriptional insights into PA biosynthesis and insolubilization during the late development stages based on the C-PCNA D. kaki genome (unpublished). Thus, we focused on PA content variation and the expression patterns of genes involved in PA biosynthesis and insolubilization. Our work has provided additional evidence on previous knowledge and a basis for further exploration of the natural deastringency of C-PCNA persimmon.

Metabolic engineering of Aureobasidium melanogenum 9–1 for overproduction of liamocins by enhancing supply of acetyl-CoA and ATP

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9–1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.

Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization

Acidic and chemical inhibitor stresses undermine efficient lactic acid bioproduction from lignocellulosic feedstock. Requisite coping treatments, such as detoxification and neutralizing agent supplementation, can be eliminated if a strong microbial host is employed in the process. Here, we exploited an originally robust yeast, Saccharomyces cerevisiae BTCC3, as a production platform for lactic acid. This wild-type strain exhibited a rapid cell growth in the presence of various chemical inhibitors compared to laboratory and industrial strains, namely BY4741 and Ethanol-red. Pathway engineering was performed on the strain by introducing an exogenous LDH gene after disrupting the PDC1 and PDC5 genes. Facilitated by this engineered strain, high cell density cultivation could generate lactic acid with productivity at 4.80 and 3.68 g L−1 h−1 under semi-neutralized and non-neutralized conditions, respectively. Those values were relatively higher compared to other studies. Cultivation using real lignocellulosic hydrolysate was conducted to assess the performance of this engineered strain. Non-neutralized fermentation using non-detoxified hydrolysate from sugarcane bagasse as a medium could produce lactic acid at 1.69 g L−1 h−1, which was competitive to the results from other reports that still included detoxification and neutralization steps in their experiments. This strategy could make the overall lactic acid bioproduction process simpler, greener, and more cost-efficient.

A gene cluster with positive and negative elements controls bistability and hysteresis of the crippled versus normal growth in the fungus Podospora anserina

The Crippled Growth (CG) cell degeneration of the model ascomycete Podospora anserina (strain S) is controlled by a prion-like element and has been linked to the self-activation of the PaMpk1 MAP kinase cascade. Here, we report on the identification of the “86–11” locus containing twelve genes, ten of which are involved either in setting up the self-activation loop of CG or in inhibiting this loop, as demonstrated by targeted gene deletion. Interestingly, deletion of the whole locus results only in the elimination of CG and in no detectable additional physiological defect. Sequence comparison shows that these ten genes belong to four different families, each one endowed with a specific activity: two encode factors activating the loop, a third one encodes a factor crucial for inhibition of the loop and the fourth one participates in inhibiting the loop in a pathway parallel to the one controlled by the previously described PDC1 gene. Intriguingly, a very distant homologue of this “86–11” locus is present at the syntenic position in Podospora comata (strain T) that do not present Crippled Growth. Introgression of the P. comata strain T locus in P. anserina strain S and the P. anserina strain S in P. comata strain T showed that both drive CG in the P. anserina strain S genetic background, but not in the genetic background of strain P. comata T, indicating that genetic determinants outside the twelve-gene locus are responsible for lack of CG in P. comata strain T. Our data question the role of this twelve-gene locus in the physiology of P. anserina.

Effectiveness of the Bacillus subtilis genome-reduced strain as an ethanol production host.

We investigated the performance of a genome-reduced strain of Bacillus subtilis MGB874, whose 0.87 Mbp of genomic DNA was cumulatively deleted, as an ethanol production host. A recombinant strain A267_EtOH was constructed by introducing the pdc and adhB genes from Zymomonas mobilis, both of which were expressed from an isopropyl-β-d-1-thiogalactopyranoside-inducible spac promoter, into the A267 strain, a tryptophan prototrophic derivative of the MGB874 with disruption of metabolic pathways for producing lactic acid, acetic acid, and acetoin. Focusing on the stationary phase in fed-batch fermentation, 1.6 g L-1 ethanol was produced by the A267_EtOH strain after 144 h. Moreover, its ethanol production further increased by approximately 3.7-fold (5.9 g L-1) at 80 h through replacing the spac promoter for expressing pdc and adhB genes with the lytR promoter and the yield was about 112%. These results indicate that the MGB874 is an effective host for ethanol production during the stationary phase.

Transcriptome analysis of immune cells from Behçet’s syndrome patients: the importance of IL-17-producing cells and antigen-presenting cells in the pathogenesis of Behçet’s syndrome

BackgroundBehçet’s syndrome (BS) is an immune-mediated disease characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin symptoms. HLA-B51, as well as other genetic polymorphisms, has been reported to be associated with BS; however, the pathogenesis of BS and its relationship to genetic risk factors still remain unclear. To address these points, we performed immunophenotyping and transcriptome analysis of immune cells from BS patients and healthy donors.MethodsImmuNexUT is a comprehensive database consisting of RNA sequencing data and eQTL database of immune cell subsets from patients with immune-mediated diseases and healthy donors, and flow cytometry data and transcriptome data from 23 BS patients and 28 healthy donors from the ImmuNexUT study were utilized for this study. Differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify genes associated with BS and clinical features of BS. eQTL database was used to assess the relationship between genetic risk factors of BS with those genes.ResultsThe frequency of Th17 cells was increased in BS patients, and transcriptome analysis of Th17 cells suggested the activation of the NFκB pathway in Th17 cells of BS patients. Next, WGCNA was used to group genes into modules with similar expression patterns in each subset. Modules of antigen-presenting cells were associated with BS, and pathway analysis suggested the activation of antigen-presenting cells of BS patients. Further examination of genes in BS-associated modules indicated that the expression of YBX3, a member of a plasmacytoid dendritic cell (pDC) gene module associated with BS, is influenced by a BS risk polymorphism, rs2617170, in pDCs, suggesting that YBX3 may be a key molecule connecting genetic risk factors of BS with disease pathogenesis. Furthermore, pathway analysis of modules associated with HLA-B51 indicated that the association of IL-17-associated pathways in memory CD8+ T cells with HLA-B51; therefore, IL-17-producing CD8+ T cells, Tc17 cells, may play a critical role in BS.ConclusionsVarious cells including CD4+ T cells, CD8+ T cells, and antigen-presenting cells are important in the pathogenesis of BS. Tc17 cells and YBX3 may be potential therapeutic targets in BS.

In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain

BackgroundThe current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification.ResultsWe have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced.ConclusionsThis work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Snorkeling Strategy: Tolerance to Flooding in Rice and Potential Application for Weed Management

Flooding is an important strategy for weed control in paddy rice fields. However, terrestrial weeds had evolved mechanisms of tolerance to flooding, resulting in new ‘snorkeling’ ecotypes. The aim of this review is to discuss the mechanisms of flooding tolerance in cultivated and weedy rice at different plant stages and the putative utility of this trait for weed management. Knowledge about flooding tolerance is derived primarily from crop models, mainly rice. The rice model informs us about the possible flooding tolerance mechanisms in weedy rice, Echinochloa species, and other weeds. During germination, the gene related to carbohydrate mobilization and energy intake (RAmy3D), and genes involved in metabolism maintenance under anoxia (ADH, PDC, and OsB12D1) are the most important for flooding tolerance. Flooding tolerance during emergence involved responses promoted by ethylene and induction of RAmy3D, ADH, PDC, and OsB12D1. Plant species tolerant to complete submersion also employ escape strategies or the ability to become quiescent during the submergence period. In weedy rice, the expression of PDC1, SUS3, and SUB1 genes is not directly related to flooding tolerance, contrary to what was learned in cultivated rice. Mitigation of flooding tolerance in weeds could be achieved with biotechnological approaches and genetic manipulation of flood tolerance genes through RNAi and transposons, providing a potential new tool for weed management.

Xylose 기질을 소모하는 재조합 Saccharomyces cerevisiae의 lactic acid 생산수율 향상을 위한 조건 탐색

Lactic acid is widely used in the food, pharmaceutical, textile and chemical industries. Also, poly-lactic acid (PLA) is in increasing demand as an environmentally friendly and biodegradable plastic. In our prior study, a lactic acid-producing, xylose-fermenting Saccharomyces cerevisiae strain (SR8 LDH) has been developed. In the present study, we tested the SR8 LDH strain under various fermentation conditions to discover the most important condition determining lactic acid and ethanol production profiles. Using xylose as a substrate significantly improved lactic acid yields compared to using glucose under microaerobic conditions. High initial cell density improved lactic acid productivity, but it did not affect ethanol production. Lastly, when fermenting xylose, the PDC1 gene encoding pyruvate decarboxylase was transcriptionally repressed, which might be associated with the production of low ethanol and high lactic acid. These results indicate that the type of a carbon source, i.e. using xylose instead of glucose could be a promising solution for lactic acid production by engineered S. cerevisiae.

Pathway Engineering for Phenethylamine Production in Escherichia coli.

In this study, the metabolic pathway of phenethylamine synthesis was reconstructed by chromosomal integration and overexpression of the Enterococcus faecium pdc gene encoding phenylalanine decarboxylase in Escherichia coli. The genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG), shikimate kinase II (aroL), chorismate mutase/prephenate dehydratase (pheA), and tyrosine aminotransferase (tyrB) in the phenethylamine synthetic pathway were sequentially chromosomally overexpressed. The phosphotransferase system was replaced by deleting the ptsH-ptsI-crr genes and chromosomally overexpressing the genes encoding galactose permease (galP) and glucokinase (glk). In addition, the zwf gene encoding glucose-6-phosphate dehydrogenase in the pentose phosphate pathway was chromosomally overexpressed, generating the final engineered E. coli strain AUD9. The AUD9 strain produced 2.65 g L-1 phenethylamine with a yield of 0.27 g of phenethylamine g-1 glucose in batch fermentation; fed-batch fermentation of AUD9 produced 38.82 g L-1 phenethylamine with a productivity of 1.08 g L-1 h-1 phenethylamine, demonstrating its potential for industrial fermentative production of phenethylamine.

Metabolic plasticity mediates differential responses to spring and autumn migrations: Evidence from gene expression patterns in migratory buntings.

What is the central question of this study? What are the molecular underpinnings of seasonal metabolic plasticity during spring and autumn migrations in songbirds? What is the main finding and its importance? We report differences in mRNA levels of genes involved in the regulation of glucose and fat metabolism between photoinduced non-migratory and migratory states and between the spring and autumn migratory states. Higher expression of genes associated with fat mobilization and energy generation in the spring than in the autumn migration suggests differential activation of the metabolic pathways or alteration in the efficiency of existing functional machinery during annual journeys between nearly fixed destinations. The molecular underpinnings of metabolic plasticity underlying differential responses to spring and autumn migrations are not well understood. We investigated this by examining the differences in mRNA levels of metabolic genes in the liver, muscle and adipose tissues of night-migratory red-headed buntings between photostimulated non-migratory and migratory states and between spring and autumn migratory states. Buntings accumulated more subcutaneous fat and hepatic lipid, had higher body mass, larger adipose cells and higher circulating triglyceride and free fatty acid levels and exhibited more intense Zugunruhe in the spring migratory state than in the autumn migratory state. More importantly, we found differences in the hepatic expression of pdc and pdk genes, indicating a differential acetyl-CoA requirement, and of the mdh and ogdh genes, suggesting differential oxidative phosphorylation between the non-migratory and migratory states and between the spring and autumn migratory states. Differences in fasn, bmal1 and glut1 mRNA levels were consistent with this and suggested seasonal differences in lipogenesis and/or glucose uptake. Likewise, differences in mRNA levels of genes coding for lipases (atgl and lpl) suggested that adipose triglycerides and free fatty acids serve largely as the metabolic substrate. Furthermore, changes in mRNA levels of genes coding for the fatty acid binding protein (fabp3) and fatty acid translocases (cd36) were consistent with differential fat fuel supply (via circulating free fatty acids) to aerobically exercising flight muscles between the spring and autumn migrations. These results show seasonal adaptation of genetic pathway(s) underlying seasonal metabolic plasticity that seems to mediate differential responses to spring and autumn migrations in latitudinal migratory songbirds.

Metabolic Engineering of a Homoserine-Derived Non-Natural Pathway for the De Novo Production of 1,3-Propanediol from Glucose.

Engineering a homoserine-derived non-natural pathway allows heterologous production of 1,3-propanediol (1,3-PDO) from glucose without adding expensive vitamin B12. Due to the lack of efficient enzymes to catalyze the deamination of homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate, the previously engineered strain can only produce 51.5 mg/L 1,3-PDO using homoserine and glucose as cosubstrates. In this study, we systematically screened the enzymes from different protein families to catalyze the two corresponding reactions and further optimized the selected enzymes by protein engineering. Together with the improvement of homoserine supply by systematic metabolic engineering, an engineered Escherichia coli strain with an optimal combination of aspartate transaminase ( aspC) from E.coli, pyruvate decarboxylase ( pdc) from Zymomonas mobiliz, and alcohol dehydrogenase yqhD from E.coli can produce 0.32 g/L 1,3-PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further increased to 0.49 g/L or 0.63 g/L by introducing a point mutation of I472A into pdc gene or constructing a fusion protein between aspC and pdc. This study lays the basis for developing a potential process for 1,3-PDO production from sugars without using expensive coenzyme B12.

Expression of Saccharomyces cerevisiae cDNAs to enhance the growth of non-ethanol-producing S. cerevisiae strains lacking pyruvate decarboxylases

Metabolic engineering of Saccharomyces cerevisiae often requires a restriction on the ethanol biosynthesis pathway. The non-ethanol-producing strains, however, are slow growers. In this study, a cDNA library constructed from S.cerevisiae was used to improve the slow growth of non-ethanol-producing S.cerevisiae strains lacking all pyruvate decarboxylase enzymes (Pdc-, YSM021). Among the obtained 120 constructs expressing cDNAs, 34 transformants showed a stable phenotype with quicker growth. Sequence analysis showed that the open reading frames of PDC1, DUG1 (Cys-Gly metallo-di-peptidase in the glutathione degradation pathway), and TEF1 (translational elongation factor EF-1 alpha) genes were inserted into the plasmids of 32, 1, and 1 engineered strains, respectively. DUG1 function was confirmed by the construction of YSM021 pGK416-DUG1 strain because the specific growth rate of YSM021 pGK416-DUG1 (0.032 ± 0.0005 h-1) was significantly higher than that of the control strains (0.029 ± 0.0008h-1). This suggested that cysteine supplied from glutathione was probably used for cell growth and for construction of Fe-S clusters. The results showed that the overexpression of cDNAs is a promising approach to engineer S.cerevisiae metabolism.

Genome-Scale In Silico Analysis for Enhanced Production of Succinic Acid in Zymomonas mobilis

Presented herein is a model-driven strategy for characterizing the production capability of expression host and subsequently identifying targets for strain improvement by resorting to network structural comparison with reference strain and in silico analysis of genome-scale metabolic model. The applicability of the strategy was demonstrated by exploring the capability of Zymomonas mobilis, as a succinic acid producer. Initially, the central metabolism of Z. mobilis was compared with reference producer, Mannheimia succiniciproducens, in order to identify gene deletion targets. It was followed by combinatorial gene deletion analysis. Remarkably, resultant in silico strains suggested that knocking out pdc, ldh, and pfl genes encoding pyruvate-consuming reactions as well as the cl gene leads to fifteen-fold increase in succinic acid molar yield. The current exploratory work could be a promising support to wet experiments by providing guidance for metabolic engineering strategies and lowering the number of trials and errors.