A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Investigation on Intestinal Proteins and Drug Metabolizing Enzymes in Simulated Microgravity Rats by a Proteomics Method.

The present study aimed to investigate the change of intestinal mucosa proteins, especially the alteration of intestinal drug metabolizing enzymes (IDMEs) following 14-day simulated microgravity. Morey-Holton tail-suspension analog was used to simulate microgravity. Intestinal mucosa proteins of rats were determined by label-free quantitative proteomic strategy. A total of 335 differentially expressed proteins (DEPs) were identified, 190 DEPs were upregulated, and 145 DEPs were downregulated. According to bioinformatic analysis, most of DEPs exhibited hydrolase, oxidoreductase, transferase, ligase, or lyase catalytic activity. DEPs were mainly enriched in metabolic pathways, including metabolism of amino acid, glucose, and carbon. Moreover, 11 of DEPs were involved in exogenous drug and xenobiotics metabolism. Owing to the importance of IDMEs for the efficacy and safety of oral drugs, the expression of cytochrome P450 1A2 (CYP1A2), CYP2D1, CYP3A2, CYP2E1, alcohol dehydrogenase 1 (ADH1), and glutathione S-transferase mu 5 (GSTM5) in rat intestine mucosa was determined by Western-blot. The activity of ADH, aldehyde dehydrogenase (ALDH) and GST was evaluated. Compared with control rats, the expression of CYP1A2, CYP2D1, CYP3A2, and ADH1 in the simulated microgravity (SMG) group of rats were dramatically decreased by 33.16%, 21.93%, 48.49%, and 22.83%, respectively. GSTM5 was significantly upregulated by 53.14% and CYP2E1 expression did not show a dramatical change in SMG group rats. Moreover, 14-day SMG reduced ADH activity, while ALDH and GST activities was not altered remarkably. It could be concluded that SMG dramatically affected the expression and activity of some IDMEs, which might alter the efficacy or safety of their substrate drugs under microgravity. The present study provided some preliminary information on IDMEs under microgravity. It revealed the potential effect of SMG on intestinal metabolism, which may be helpful to understand the intestinal health of astronauts and medication use.

Expression of tandem gene duplicates is often greater than twofold.

Tandem gene duplication is an important mutational process in evolutionary adaptation and human disease. Hypothetically, two tandem gene copies should produce twice the output of a single gene, but this expectation has not been rigorously investigated. Here, we show that tandem duplication often results in more than double the gene activity. A naturally occurring tandem duplication of the Alcohol dehydrogenase (Adh) gene exhibits 2.6-fold greater expression than the single-copy gene in transgenic Drosophila This tandem duplication also exhibits greater activity than two copies of the gene in trans, demonstrating that it is the tandem arrangement and not copy number that is the cause of overactivity. We also show that tandem duplication of an unrelated synthetic reporter gene is overactive (2.3- to 5.1-fold) at all sites in the genome that we tested, suggesting that overactivity could be a general property of tandem gene duplicates. Overactivity occurs at the level of RNA transcription, and therefore tandem duplicate overactivity appears to be a previously unidentified form of position effect. The increment of surplus gene expression observed is comparable to many regulatory mutations fixed in nature and, if typical of other genomes, would shape the fate of tandem duplicates in evolution.

Increased GSNOR Expression during Aging Impairs Cognitive Function and Decreases S-Nitrosation of CaMKIIα.

As the population ages, an increasing number of people suffer from age-related cognitive impairment. However, the mechanisms underlying this process remain unclear. Here, we found that S-nitrosoglutathione reductase (GSNOR), the key enzyme that metabolizes intracellular nitric oxide (NO) and regulates S-nitrosation, was significantly increased in the hippocampus of both aging humans and mice. Transgenic mice overexpressing GSNOR exclusively in neurons showed cognitive impairment in behavioral tests, including the Morris water maze, fear conditioning, and the Y-maze test. We also found that GSNOR transgenic mice have LTP defects and lower dendrite spine density, whereas GSNOR knock-out mice rescued the age-related cognitive impairment. Analysis of S-nitrosation showed significantly decreased hippocampal CaMKIIα S-nitrosation in naturally aged mice and GSNOR transgenic mice. Consistent with the change in CaMKIIα S-nitrosation, the accumulation of CaMKIIα in the hippocampal synaptosomal fraction, as well as its downstream signaling targets p(S831)-GLUR1, was also significantly decreased. All these effects could be rescued in the GSNOR knock-out mice. We further verified that the S-nitrosation of CaMKIIα was responsible for the CaMKIIα synaptosomal accumulation by mutating CaMKIIα S-nitrosated sites (C280/C289). Upregulation of the NO signaling pathway rescued the cognitive impairment in GSNOR transgenic mice. In summary, our research demonstrates that GSNOR impairs cognitive function in aging and it could serve as a new potential target for the treatment of age-related cognitive impairment. In contrast to the free radical theory of aging, NO signaling deficiency may be the main mediator of age-related cognitive impairment.SIGNIFICANCE STATEMENT This study indicated that S-nitrosoglutathione reductase (GSNOR), a key protein S-nitrosation metabolic enzyme, is a new potential target in age-related cognitive impairment; and in contrast to the free radical theory of aging, NO signaling deficiency may be the main cause of this process. In addition, increased GSNOR expression during aging decreases S-nitrosation of CaMKIIα and reduces CaMKIIα synaptosomal accumulation. To our knowledge, it is for the first time to show the cellular function regulation of CaMKIIα by GSNOR-dependent S-nitrosation as a new post-translational modification after its phosphorylation was explored. These findings elucidate a novel mechanism of age-related cognitive impairment and may provide a new potential target and strategy for slowing down this process.

De novo synthesis of the sedative valerenic acid in Saccharomyces cerevisiae.

Valeriana officinalis (Valerian) root extracts have been used by European and Asian cultures for millennia for their anxiolytic and sedative properties. However, the efficacy of these extracts suffers from variable yields and composition, making these extracts a prime candidate for microbial production. Recently, valerenic acid, a C15 sesquiterpenoid, was identified as the active compound that modulates the GABAA channel. Although the first committed step, valerena-4,7(11)-diene synthase, has been identified and described, the complete valerenic acid biosynthetic pathway remains to be elucidated. Sequence homology and tissue-specific expression profiles of V. officinalis putative P450s led to the discovery of a V. officinalis valerena-4,7(11)-diene oxidase, VoCYP71DJ1, which required coexpression with a V. officinalis alcohol dehydrogenase and aldehyde dehydrogenase to complete valerenic acid biosynthesis in yeast. Further, we demonstrated the stable integration of all pathway enzymes in yeast, resulting in the production of 140 mg/L of valerena-4,7(11)-diene and 4 mg/L of valerenic acid in milliliter plates. These findings showcase Saccharomyces cerevisiae's potential as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, possibly paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines.

Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Alcohol Dehydrogenases and Acetaldehyde Dehydrogenases are Beneficial for Decidual Stromal Cells to Resist the Damage from Alcohol.

Aims: The aim of this study was to examine the effect of alcohol on the decidualization of human endometrial stromal cells during early pregnancy. Methods: During in vitro decidualization, human endometrial stromal cells were treated with alcohol, 4-methylpyrazole hydrochloride (FPZ), the inhibitor of alcohol dehydrogenases (ADHs), and tetraethylthiuram disulfide (DSF), the inhibitor of acetaldehyde dehydrogenases (ALDHs), respectively. Cell viability and decidualization were examined. Apoptosis and proliferation were also evaluated. Results: The findings showed that ADHs and ALDHs were up-regulated during decidualization. After alcohol treatment, the cell viability of decidual stromal cells was significantly higher than control, which was abrogated by FPZ or DSF. When cells were treated with alcohol, proliferation-related signal pathways were up-regulated in decidualized cells. Additionally, FOXO1 transcriptionally up-regulates ADH1B. Conclusion: Our study provided an evidence that highly expressed ADHs and ALDHs endow decidual stromal cells an ability to alleviate the harm from alcohol.

Promoter sequence determines the relationship between expression level and noise.

The ability of cells to accurately control gene expression levels in response to extracellular cues is limited by the inherently stochastic nature of transcriptional regulation. A change in transcription factor (TF) activity results in changes in the expression of its targets, but the way in which cell-to-cell variability in expression (noise) changes as a function of TF activity, and whether targets of the same TF behave similarly, is not known. Here, we measure expression and noise as a function of TF activity for 16 native targets of the transcription factor Zap1 that are regulated by it through diverse mechanisms. For most activated and repressed Zap1 targets, noise decreases as expression increases. Kinetic modeling suggests that this is due to two distinct Zap1-mediated mechanisms that both change the frequency of transcriptional bursts. Notably, we found that another mechanism of repression by Zap1, which is encoded in the promoter DNA, likely decreases the size of transcriptional bursts, producing a unique transcriptional state characterized by low expression and low noise. In addition, we find that further reduction in noise is achieved when a single TF both activates and represses a single target gene. Our results suggest a global principle whereby at low TF concentrations, the dominant source of differences in expression between promoters stems from differences in burst frequency, whereas at high TF concentrations differences in burst size dominate. Taken together, we show that the precise amount by which noise changes with expression is specific to the regulatory mechanism of transcription and translation that acts at each gene.

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor.

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

A whole cell biocatalyst for double oxidation of cyclooctane.

A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.

Expression profile of rice Hsp genes under anoxic stress.

Although flooding is one of the most important environmental stresses worldwide, not all plant species are intolerant to its effects. Species from semi-aquatic environments, such as rice, have the capacity to cope with flooding stress. Heat-shock proteins (Hsps) are thought to contribute to cellular homeostasis under both optimal and adverse growth conditions. Studies of gene expression in plants exposed to low levels of oxygen revealed the up-regulation of Hsp genes. However, it is not clear whether Hsp genes are transcribed as a function of tolerance or whether they represent a response to anoxic stress. Therefore, the accumulation of Hsp gene transcripts was investigated in two different cultivars, "Nipponbare" (flooding tolerant) and "IPSL 2070" (flooding sensitive), subjected to anoxic stress. Fifteen-day-old rice root seedlings from both cultivars were used. Four different treatments were performed: no anoxia (control); 24-h anoxia; 48-h anoxia; and 72-h anoxia. Anoxic stress was confirmed by the increased gene expression of alcohol dehydrogenase. The data obtained showed that both rice cultivars ("Nipponbare" and "IPSL 2070") accumulated Hsp gene transcripts under anoxic stress; however, the majority of the Hsp genes evaluated were responsive to anoxic stress in "IPSL 2070" (flooding sensitive), whereas in "Nipponbare" (flooding tolerant), only six genes were highly up-regulated. This suggests that although Hsps have an important role in the response to anoxia, they are not the major cause of tolerance.

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR-XDH pathway.

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD(+) to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.

Efficient production of 1,3-propanediol from glycerol upon constitutive expression of the 1,3-propanediol oxidoreductase gene in engineered Klebsiella pneumoniae with elimination of by-product formation.

In the present study, we developed an efficient method of 1,3-propanediol (1,3-PD) production from glycerol by genetic engineering of Klebsiella pneumoniae AK mutant strains. The proposed approach eliminated by-product formation and IPTG induction resulted in maximal production of 1,3-PD. A series of recombinant strains was designed to constitutively express the dhaB and/or dhaT genes, using the bacteriophage T5 P(DE20) promoter and the rho-independent transcription termination signal of the Rahnella aquatilis levansucrase gene. Among these strains, AK/pConT expressing dhaT alone gave the highest yield of 1,3-PD. Fed-batch fermentation resulted in efficient production of 1,3-PD from either pure or crude glycerol, without by-product formation.

[Cloning, expression and characterization of alcohol dehydrogenase and aldehyde dehydrogenase from Bacillus pseudofirmus OF4].

OBJECTIVE: To clone and characterize the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from Bacillus pseudofirmus OF4. METHODS: Genes of adh and aldh were cloned by PCR; expression vectors pET-Ahd and pET-Aldh were constructed and expressed in Escherichia coli BL21 (DE3). After Ni-NTA column chromatography purification, the protein was characterized. RESULTS: The optimal temperature and pH of ALDH was 35 degrees C and 8.0, the specific activities of ALDH was 979.6 U/mg protein, the thermostability at 25 degrees C and 35 degrees C was better than at 45 degrees C. Although the expression level of ADH was too low to purify, but it was found that ADH had high catalytic activities by experiments of co-expression and ethanol tolerance. CONCLUSION: Adh and aldh from B. pseudofirmus OF4 were cloned successfully. Co-expression of double genes could greatly increase the host strain on ethanol tolerance.

Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human small intestine.

BACKGROUND: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol (EtOH). Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. This study aimed to systematically determine the functional expressions and cellular localization of ADH and ALDH family members in human small bowel. METHODS: One hundred and seventeen surgical specimens of duodenal mucosae, 34 jejunal mucosal specimens, and 14 paired specimens of stomach, duodenum, and jejunum from same individuals were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing, and the ADH/ALDH activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies, and the cellular localizations were detected by immunohistochemistry and histochemistry. RESULTS: The activities of ADH1C*1/*1 allelotype were significantly higher than those of the ADH1C*1/*2 allelotype in duodenum (p < 0.001) and in jejunum (p < 0.05); and the activity of ADH2-expressing phenotype was significantly higher than that of the ADH2-missing phenotype in duodenum (p < 0.05). The activities of ALDH2-inactive phenotype were not significantly different from those of the ALDH2-active phenotype in duodenum and jejunum. Stomach exhibited significantly lower ADH activity (p < 0.05), and duodenum displayed significantly lower ALDH activity (p < 0.001) comparing the paired gastric, duodenal, and jejunal mucosae of same individuals. Gender and age did not significantly influence the ADH and ALDH activities in duodenum. The isozyme protein contents in duodenum and jejunum were in the following decreasing order: ALDH1A1, ADH1/ALDH2, ADH3, ADH2, and ALDH3A1. Villous epithelial cells, cryptic Paneth cells, and Brunner's gland ductal cells revealed a greater immunostaining intensity with ADH1, ALDH1A1, and ALDH2. CONCLUSIONS: ADH and ALDH isozymes are differentially expressed in the various cell types of duodenum and jejunum. The results suggest that proximal small intestine can substantively contribute to first-pass metabolism of EtOH under certain conditions and that cytotoxic acetaldehyde and EtOH perturbation of retinol metabolism might play an etiological role in the pathogenesis of small bowel.

Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor®2 transformation/expression platform.

The Xplor®2 transformation/expression platform was employed for comparative assessment of three different yeast species as hosts for synthesis of a thermostable nicotinamide adenine dinucleotide (NAD⁺)-dependent medium-chain alcohol dehydrogenase from Rhodococcus ruber strain 219. Using yeast ribosomal DNA (rDNA) integrative expression cassettes (YRCs) and yeast integrative expression cassettes (YICs) equipped with a selection-marker module and one, two or four expression modules for transformation of auxotrophic Arxula adeninivorans, Hansenula polymorpha, and Saccharomyces cerevisiae strains, quantitative comparison of the yield of recombinant alcohol dehydrogenase RR-ADH6Hp in all three species was carried out. In all cases, the RR-ADH6H gene was expressed under the control of the strong constitutive A. adeninivorans-derived TEF1 promoter, which functions in all yeast species analyzed. Recombinant RR-ADH6Hp accumulated intracellularly in all strains tested. The best yields of active enzyme were obtained from A. adeninivorans, with S. cerevisiae producing intermediate amounts. Although H. polymorpha was the least efficient producer overall, the product obtained was most similar to the enzyme synthesized by R. ruber 219 with respect to its thermostability.

Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway.

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C(4)) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33g/l, resulting in an ethanol to glucose yield of 0.38g/g.