Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl ß- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5' untranslated region (5' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

PsrA is a novel regulator contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in Serratia marcescens.

Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.

Two-color fluorescent proteins reporting survivin regulation in breast cancer cells for high throughput drug screening.

Reporter gene assay is widely used for high throughput drug screening and drug action mechanism evaluation. In this study, we developed a robust dual-fluorescent reporter assay to detect drugs repressing the transcription of survivin, a cancer biomarker from the inhibitor of apoptosis family, in breast cancer cells cultured in three-dimensional (3D) microbioreactors. Survivin is overexpressed in numerous malignancies but almost silent in normal tissue cells and is considered a lead target for cancer therapy. Breast cancer MCF-7 cells were engineered to express enhanced green fluorescent protein driven by a survivin promoter and red fluorescent protein driven by a cytomegalovirus promoter as internal control to detect changes in survivin expression in cells as affected by drugs. This 3D dual-fluorescent reporter assay was validated with YM155 and doxorubicin, which were known to downregulate survivin in cancer cells, and further evaluated with two widely used anticancer compounds, cisplatin, and epigallocatechin gallate, to evaluate their effects on survivin expression. The results showed that the 3D dual-fluorescent reporter assay was robust for high throughput screening of drugs targeting survivin in breast cancer cells.

High-rate continuous n-butanol production by Clostridium acetobutylicum from glucose and butyric acid in a single-pass fibrous-bed bioreactor.

Biobutanol produced in acetone-butanol-ethanol (ABE) fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs, but are rarely used in industrial fermentation because of increased risks of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At a dilution rate of 1.88 h-1 , butanol was produced at 9.55 g/L, with a yield of 0.24 g/g and productivity of 16.8 g/L/h, which was the highest productivity ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over 1 month.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Enhanced Prodigiosin Production in Serratia marcescens JNB5-1 by Introduction of a Polynucleotide Fragment into the pigN 3' Untranslated Region and Disulfide Bonds into O-Methyl Transferase (PigF).

In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.

Regulator RcsB Controls Prodigiosin Synthesis and Various Cellular Processes in Serratia marcescens JNB5-1.

Prodigiosin (PG), a red linear tripyrrole pigment normally secreted by Serratia marcescens, has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene BVG90_13250 (rcsB) negatively controlled prodigiosin biosynthesis in S. marcescens Disruption of rcsB conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated pig operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in S. marcescens Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in S. marcescens and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in S. marcescensIMPORTANCE RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in Enterobacteriaceae However, information on the function of the RcsB protein in bacteria, especially in S. marcescens, remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in S. marcescens, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in S. marcescens.

Sustainable production and biomedical application of polymalic acid from renewable biomass and food processing wastes.

Polymalic acid (PMA), a homopolymer of L-malic acid (MA) generated from a yeast-like fungus Aureobasidium pullulans, has unique properties and many applications in food, biomedical, and environmental fields. Acid hydrolysis of PMA, releasing the monomer MA, has become a novel process for the production of bio-based MA, which currently is produced by chemical synthesis using petroleum-derived feedstocks. Recently, current researches attempted to develop economically competitive process for PMA and MA production from renewable biomass feedstocks. Compared to lignocellulosic biomass, PMA and MA production from low-value food processing wastes or by-products, generated from corn, sugarcane, or soybean refinery industries, showed more economical and sustainable for developing a MA derivatives platform from biomass biorefinery to chemical conversion. In the review, we compared the process feasibility for PMA fermentation with lignocellulosic biomass and food process wastes. Some useful strategies for metabolic engineering are summarized. Its changeable applicability and future prospects in food and biomedical fields are also discussed.

Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 µM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 µM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 µM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlACA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnrCA -thlACA -hbdCT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ß-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.

A novel ß-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose.

Galacto-oligosaccharides (GOS), which can be produced by enzymatic transgalactosylation of lactose with ß-galactosidases, have attracted much attention in recent years because of their prebiotic functions and wide uses in infant formula, infant foods, livestock feed, and pet food industries. In this study, a novel ß-galactosidase-producing Klebsiella oxytoca ZJUH1705, identified by its 16S rRNA sequence (GenBank accession no. MH981243), was isolated. Two ß-galactosidase genes, bga 1 encoding a 2058-bp fragment (GenBank accession no. MH986613) and bga 2 encoding a 3108-bp fragment (GenBank accession no. MN182756), were cloned from K. oxytoca ZJUH1705 and expressed in E. coli. The purified ß-gal 1 and ß-gal 2 had the specific activity of 217.56 U mg-1 and 57.9 U mg-1, respectively, at the optimal pH of 7.0. The reaction kinetic parameters Km, Vmax, and Kcat with oNPG as the substrate at 40 °C were 5.62 mM, 167.1 µmol mg-1 min-1, and 218.1 s-1, respectively, for ß-gal 1 and 3.91 mM, 14.6 µmol mg-1 min-1, and 28.9 s-1, respectively, for ß-gal 2. Although ß-gal 1 had a higher enzyme activity for lactose hydrolysis, only ß-gal 2 had a high transgalactosylation capacity. Using ß-gal 2 with the addition ratio of ~ 2.5 U g-1 lactose, a high GOS yield of 45.5 ± 2.3% (w/w-1) was obtained from lactose (40% w/w-1 or 480 g L-1) in a phosphate buffer (100 mM, pH 7.0) at 40 °C in 48 h. Thus, the ß-gal 2 from K. oxytoca ZJUH1705 would be a promising biocatalyst for GOS production from lactose.Key Points• A novel bacterial ß-galactosidase producer was isolated and identified.• ß-Galactosidases were cloned and expressed in heterologous strain and characterized.• Both enzymes have hydrolytic activity but only one have transglycosilation activity.• The developed process with ß-gal 2 could provide an alternative for GOS production.

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies.

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO2. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H+-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na+-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.

Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum.

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30 g/g and titer of > 20 g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.

Biosynthesis of polymalic acid in fermentation: advances and prospects for industrial application.

Some microorganisms naturally produce ß-poly(l-malic acid) (PMA), which has excellent water solubility, biodegradability, and biocompatibility properties. PMA has broad prospective applications as novel biopolymeric materials and carriers in the drug, food, and biomedical fields. Malic acid, a four-carbon dicarboxylic acid, is widely used in foods and pharmaceuticals, as a platform chemical. Currently, malic acid produced through chemical synthesis and is available as a racemic mixture of l- and d-forms. The d-form malic acid exhibits safety concerns for human consumption. There is extensive interest to develop economical bioprocesses for l-malic acid and PMA production from renewable biomass feedstocks. In this review, we focus on PMA biosynthesis by Aureobasidium pullulans, a black yeast with a large genome containing genes encoding many hydrolases capable of degrading various plant materials. The metabolic and regulatory pathways for PMA biosynthesis, metabolic engineering strategies for strain development, process factors affecting fermentation kinetics and PMA production, and downstream processing for PMA recovery and purification are discussed. Prospects of microbial PMA and malic acid production are also considered.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.

Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440.

BACKGROUND: Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. RESULTS: In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. CONCLUSIONS: This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site.

Engineering Clostridium for improved solvent production: recent progress and perspective.

Clostridia are Gram-positive, spore-forming, obligate anaerobic bacteria that can produce solvents such as acetone, ethanol, and butanol, which can be used as biofuels or building block chemicals. Many successful attempts have been made to improve solvent yield and titer from sugars through metabolic engineering of solventogenic and acidogenic clostridia. More recently, cellulolytic and acetogenic clostridia have also attracted high interests for their ability to utilize low-cost renewable substrates such as cellulose and syngas. Process engineering such as in situ butanol recovery and consolidated bioprocessing (CBP) has been developed for improved solvent titer and productivity. This review focuses on metabolic and process engineering strategies for solvent production from sugars, lignocellulosic biomass, and syngas by various clostridia, including conventional solventogenic Clostridium acetobutylicum, engineered acidogens such as C. tyrobutyricum and C. cellulovorans, and carboxydotrophic acetogens such as C. carboxidivorans and C. ljungdahlii.

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans.

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

Identification of steroid C27 monooxygenase isoenzymes involved in sterol catabolism and stepwise pathway engineering of Mycobacterium neoaurum for improved androst-1,4-diene-3,17-dione production.

Cholesterol oxidase, steroid C27 monooxygenase and 3-ketosteroid-Δ1-dehydrogenase are key enzymes involved in microbial catabolism of sterols. Here, three isoenzymes of steroid C27 monooxygenase were firstly characterized from Mycobacterium neoaurum as the key enzyme in sterol C27-hydroxylation. Among these three isoenzymes, steroid C27 monooxygenase 2 exhibits the strongest function in sterol catabolism. To improve androst-1,4-diene-3,17-dione production, cholesterol oxidase, steroid C27 monooxygenase 2 and 3-ketosteroid-Δ1-dehydrogenase were coexpressed to strengthen the metabolic flux to androst-1,4-diene-3,17-dione, and 3-ketosteroid 9α-hydroxylase, which catalyzes the androst-1,4-diene-3,17-dione catabolism, was disrupted to block the androst-1,4-diene-3,17-dione degradation pathway in M. neoaurum JC-12. Finally, the recombinant strain JC-12S2-choM-ksdd/ΔkshA produced 20.1 g/L androst-1,4-diene-3,17-dione, which is the highest reported production with sterols as substrate. Therefore, this work is hopes to pave the way for efficient androst-1,4-diene-3,17-dione production through metabolic engineering.