Multicolor Chromism from a Single Chromophore through Synergistic Coupling of Mechanochromic and Photochromic Subunits.

We report a versatile mechanophore exhibiting a vividly detectable, light-regulable multicolor mechanochromism. Such optical features rely on the synergistic coupling of mechanochromic bis-rhodamine (Rh) and photochromic bisthienylethene (BTE). Poly(methyl acrylate)s incorporating this bis-mechanophore can be mechanically activated under sonication. The relative distribution of the two distinctly colored and fluorescent Rh ring-opening products is altered with different magnitudes of applied force. Orthogonal use of the photochromic reaction of the BTE core can strengthen the mechanochromism and gate the mechanofluorescence in polymers. Due to increased conjugation offered by the BTE linker, both force- and light-induced optical signals display high contrast. Combined DFT simulated and experimental results reveal that the three subunits (two Rhs and one BTE) in this chromophore are activated sequentially, thus generating switchable three-colored forms and gradient optical responses.

Combined enhancement of the propionyl-CoA metabolic pathway for efficient androstenedione production in Mycolicibacterium neoaurum.

BACKGROUND: The production of androstenedione (AD) from phytosterols by Mycolicibacterium neoaurum is a multi-step biotransformation process, which requires degradation of sterol side chains, accompanied by the production of propionyl-CoA. However, the transient production of large amounts of propionyl-CoA can accumulate intracellularly to produce toxic effects and severely inhibit AD production. RESULTS: In the present study, the intracellular propionyl-CoA concentration was effectively reduced and the productivity of the strain was improved by enhancing the cytosolic methyl-branched lipid synthesis pathway and increasing the expression level of nat operator gene, respectively. Subsequently, the application of a pathway combination strategy, combined and the inducible regulation strategy, further improved AD productivity with a maximum AD conversion rate of 96.88%, an increase of 13.93% over the original strain. CONCLUSIONS: Overall, we provide a new strategy for reducing propionyl-CoA stress during biotransformation for the production of AD and other steroidal drugs using phytosterols.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059 g/L/h, with a conversion of 94.41% at 24 h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20 h) with high substrate (85 g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85 g/L CA) and high transformation rate craft (94.34% within 20 h) were built.

Efficient One-Step Biocatalytic Multienzyme Cascade Strategy for Direct Conversion of Phytosterol to C-17-Hydroxylated Steroids.

Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, and boldenone (BD) is one of the important C-17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenges of the complex transformation process, high substrate costs, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17ß-Hydroxysteroid dehydrogenase (17ßHSD) and its native electron donor play significant roles in the 17ß-carbonyl reduction reaction of steroids. In this study, we bridged 17ßHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for the efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with the coexpression of 17ßhsd and the glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum. With the establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP+] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M-ayr1S.c) to 78% (in MNR M3M-ayr1&g6p with glucose supplementation), and the productivity was increased by 6.5-fold. Furthermore, under optimal glucose supplementation conditions, the yield of BD reached 82%, which is the highest yield reported for transformation from PS in one step. This study demonstrated an excellent strategy for the production of many other valuable carbonyl reduction steroidal products from natural inexpensive raw materials. IMPORTANCE Steroid C-17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for the further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residues of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicine production by Mycobacterium. This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for the direct conversion of phytosterol to C-17-hydroxylated steroids by bridging 17ß-hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum. This work has practical value for the production of many valuable hydroxylated steroids from natural inexpensive raw materials.

Engineering a Highly Biomimetic Chitosan-Based Cartilage Scaffold by Using Short Fibers and a Cartilage-Decellularized Matrix.

Engineering scaffolds with structurally and biochemically biomimicking cues is essential for the success of tissue-engineered cartilage. Chitosan (CS)-based scaffolds have been widely used for cartilage regeneration due to its chemostructural similarity to the glycosaminoglycans (GAGs) found in the extracellular matrix of cartilage. However, the weak mechanical properties and inadequate chondroinduction capacity of CS give rise to compromised efficacy of cartilage regeneration. In this study, we incorporated short fiber segments, processed from electrospun aligned poly(lactic-co-glycolic acid) (PLGA) fiber arrays, into a citric acid-modified chitosan (CC) hydrogel scaffold for mechanical strengthening and structural biomimicking and meanwhile introduced cartilage-decellularized matrix (CDM) for biochemical signaling to promote the chondroinduction activity. We found that the incorporation of PLGA short fibers and CDM remarkably strengthened the mechanical properties of the CC hydrogel (+349% in compressive strength and +153% in Young's modulus), which also exhibited a large pore size, appropriate porosity, and fast water absorption ability. Biologically, the engineered CDM-Fib/CC scaffold significantly promoted the adhesion and proliferation of chondrocytes and supported the formation of matured cartilage tissue with a cartilagelike structure and deposition of abundant cartilage ECM-specific GAGs and type II collagen (+42% in GAGs content and +295% in type II collagen content). The enhanced mechanical competency and chondroinduction capacity with the engineered CDM-Fib/CC scaffold eventually fulfilled successful in situ osteochondral regeneration in a rabbit model. This study thereby demonstrated a great potential of the engineered highly biomimetic chitosan-based scaffold in cartilage tissue repair and regeneration.

The Sterol Carrier Hydroxypropyl-ß-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum.

Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-ß-CD (HP-ß-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-ß-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-ß-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-ß-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism.IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-ß-CD (HP-ß-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum.

BACKGROUND: Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess. RESULTS: There is a complete MCC in Mycobacterium neoaurum (MNR), prpC, prpD and prpB in the prp operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of prp operon. After the overexpression of prpDCB and prpR in MNR, the significantly improved transcription levels of prpC, prpD and prpB were observed. The highest conversion ratios of AD obtained by MNR-prpDBC and MNR-prpR increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed prp operon transcription in a nitrogen-limited medium. The glnR deletion enhanced the transcription level of prpDBC and the biotransformation ability of MNR. MNR-prpR/ΔglnR was constructed by the overexpression of prpR in the glnR-deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR-prpR/ΔglnR was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR. CONCLUSION: Improvement in phytosterol biotransformation after the enhancement of propionyl-CoA metabolism through the combined modifications of the prp operon and glnR of mycobacteria was investigated for the first time. The overexpress of prpR in MNR can increase the transcription of essential genes (prpC, prpD and prpB) of MCC, reduce the intracellular propionyl-CoA level and improve bacterial viability. The knockout of glnR can enhance the adaptability of MNR to the nitrogen source. In the MNRΔglnR strain, overexpress of prpR can achieve efficient production of AD at low nitrogen levels, thus reducing the production cost. This strategy provides a reference for the economic and effective production of other valuable steroid metabolites from phytosterol in the pharmaceutical industry.

Compatible solutes adaptive alterations in Arthrobacter simplex during exposure to ethanol, and the effect of trehalose on the stress resistance and biotransformation performance.

Ethanol-tolerant Arthrobacter simplex is desirable since ethanol facilitates hydrophobic substrates dissolution on an industrial scale. Herein, alterations in compatible solutes were investigated under ethanol stress. The results showed that the amount of trehalose and glycerol increased while that of glutamate and proline decreased. The trehalose protectant role was verified and its concentration was positively related to the degree of cell tolerance. otsA, otsB and treS, three trehalose biosynthesis genes in A. simplex, also enhanced Escherichia coli stress tolerance, but the increased tolerance was dependent on the type and level of the stress. A. simplex strains accumulating trehalose showed a higher productivity in systems containing more ethanol and substrate because of better viability. The underlying mechanisms of trehalose were involved in better cell integrity, higher membrane stability, stronger reactive oxygen species scavenging capacity and higher energy level. Therefore, trehalose was a general protectant and the upregulation of its biosynthesis by genetic modification enhanced cell stress tolerance, consequently promoted productivity.

Highly efficient synthesis of boldenone from androst-4-ene-3,17-dione by Arthrobacter simplex and Pichia pastoris ordered biotransformation.

Boldenone (BD) is an important steroid hormone drug which is the derivative of testosterone. In this study, an ordered biotransformation method was proposed employing Arthrobacter simplex and recombinant Pichia pastoris with 17ß-hydroxysteroid dehydrogenase from Saccharomyces cerevisiae to produce BD from androst-4-ene-3,17-dione (AD) efficiently. To lower the oxidation towards BD in A. simplex, the transformation was conducted sequentially by C1,2 dehydrogenation in A. simplex and 17ß-carbonyl reduction in recombinant P. pastoris GS115. Moreover, the reaction system was inactivated before recombinant P. pastoris GS115 was added to further inhibit the oxidation of BD by A. simplex, and the productivity of BD was improved 10.6% compared with the control. Furthermore, by optimizing the conditions of transformation from AD to BD, 4.2 g/L BD was generated with 83% productivity from 5.0 g/L AD, which was the highest productivity reported by biological method. This study offers a promising method to produce BD by ordered biotransformation system, which can also be used to manufacture other steroidal compounds that are difficult to acquire directly.

The ethanol-induced global alteration in Arthrobacter simplex and its mutants with enhanced ethanol tolerance.

Arthrobacter simplex has received considerable interests due to its superior Δ1-dehydrogenation ability. Ethanol used as co-solvent is a stress commonly encountered during biotransformation. Therefore, studies of ethanol tolerance of A. simplex are of great importance to improve the biotransformation efficiency. In this paper, the combined analysis of physiological properties, cell compositions, stress-responsive metabolites, and proteome profiles was carried out to achieve a global view of ethanol tolerance of A. simplex. Under sublethal conditions, cell permeability and membrane fluidity exhibited concentration-dependent increase by affecting the contents or compositions of cell peptidoglycan, lipids, phospholipids, and fatty acids. Among them, cis-trans isomerization of unsaturated fatty acids was a short-term and reversible process, while the changes in phospholipid headgroups and increase in saturation degree of fatty acids were long-term and irreversible processes, which collectively counteracted the elevated membrane fluidity caused by ethanol and maintained the membrane stability. The decreased intracellular ATP content was observed at high ethanol concentration since proton motive force responsible for driving ATP synthesis was dissipated. The involvement of trehalose and glycerol, oxidative response, and DNA damage were implicated due to their changes in positive proportion to ethanol concentration. Proteomic data supported that ethanol invoked a global alteration, among which, the change patterns of proteins participated in the biosynthesis of cell wall and membrane, energy metabolism, compatible solute metabolism, and general stress response were consistent with observations from cell compositions and stress-responsive metabolites. The protective role of proteins participated in DNA repair and antioxidant system under ethanol stress was validated by overexpression of the related genes. This is the first demonstration on ethanol tolerance mechanism of A. simplex, and the current studies also provide targets to engineer ethanol tolerance of A. simplex.

Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation.

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

BACKGROUND: Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. RESULTS: Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD+/NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols. CONCLUSIONS: The ratio of NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

A new technique for promoting cyclic utilization of cyclodextrins in biotransformation.

Cyclodextrins (CDs) can improve the productivity of steroid biotransformation by enhancing substrate solubility. CDs can be recycled by grafting them with appropriate carriers. Loofah fiber is an excellent grafting material for CDs, and can be applied to the biotransformation and recycling of ß-cyclodextrin (ß-CD). In this work, a technique for recycling ß-CD in cortisone acetate (CA) biotransformation by Arthrobacter simplex CPCC 140451 was studied. Loofah fiber-grafted ß-CD (LF-ß-CD) was prepared using epichlorohydrin, which is a cross-linking agent. The grafting yield of ß-CD was 74.8 mg g-1 dried fibers. LF-ß-CD could increase the solubility of CA and enhance biotransformation. The initial conversion rate of CA was 1.5-fold higher than that of the blank group. LF-ß-CD was also used in biocatalytic reactions for eight cycles, and it maintained the conversion ratio of CA at approximately 90%. Given the above positive results, LF-ß-CD can be utilized in biotechnological recycling applications. This method can also be applied to CD derivatives and hydrophobic compounds.

Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum.

3-Ketosteroid-Δ1-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.

Effects of two kinds of imidazolium-based ionic liquids on the characteristics of steroid-transformation Arthrobacter simplex.

BACKGROUND: Ionic liquids (ILs) are a promising alternative for organic solvents because these liquids exhibit unique properties and enhanced steroid 1-dehydrogenation biotransformation caused by Arthrobacter simplex CPCC 140451 (ASP). However, the effect of ILs on the whole cell itself remains poorly understood and must be further investigated. RESULTS: A comparative investigation was performed to determine the effect of imidazolium-based ILs, namely, hydrophobic [PrMIm]PF6, and hydrophilic [PrMIm]BF4, on the steroid conversion, activity, permeability, and material basis of ASP cells. Both ILs weakened permeability barriers, enhanced steroid transformation, whereas reduced the activity of cells. The influence of [PrMIm]PF6 on the steroid conversion, permeability and activity of cells is more serious than that of [PrMIm]BF4 Transmission electron microscopy micrographs directly showed wrinkles, gross creases, and several small pores in ILs-treated cells surface. The total lipid content of [PrMIm]BF4-treated cells reduced by 8.3 %, while that of [PrMIm]PF6-treated cells reduced twice more, among which the content of long-chain fatty acids was decreased, whereas the content of unsaturated fatty acids was increased. The protein profile of LC-MS/MS revealed that the reduced proteins of cells treated with the two ILs were mainly located in the cytoplasm and plasma membrane, 19.27 % of reduced proteins were located on the cell membrane for [PrMIm]PF6-pretreated cells, whereas only 12.8 % for [PrMIm]BF4-pretreated cells. It suggests that most reduced proteins functioned in energy production and conversion, material transport and metabolism, signal recognition and transmission, transcription, and translation and posttranslational modification. In particular, the identified differential proteins functioned in the pentose phosphate pathway, synthesis of purines and pyrimidines, and oxidative phosphorylation and fatty acid pathway. CONCLUSION: Treatment with ILs improved permeability at the molecular level and exerted significant positive effects on steroid conversion. This study provides a material basis and elucidates the mechanisms underlying cellular changes that enhanced conversion rate.

Cyclic utilization of HP-ß-CD in the bioconversion of cortisone acetate by Arthrobacter simplex.

OBJECTIVE: To establish a method for the recovery and reutilization of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to lower the cost of its industrial application in cortisone acetate bioconversion. RESULTS: HP-ß-CD is not degraded by Arthrobacter simplex CPCC140451 (ASP) resting cells and 96.4 % HP-ß-CD could be recovered by isobutyl acetate extraction. Moreover, the inclusion ability of recovered HP-ß-CD barely decreased. The saccharide metabolic and catalytic activities of ASP were greater in the aqueous phase after extracting with isobutyl acetate than other organic solvents. Cyclic utilization tests showed that cortisone acetate conversion ratio was 91.0 % after eight cycles and reached 95.7 % with 0.2-0.6 mM HP-ß-CD. Furthermore, >90 % conversion ratio was reached per cycle through a co-cyclic-utilization method with HP-ß-CD and immobilized ASP. CONCLUSION: Cortisone acetate conversion ratio in the HP-ß-CD cyclic-utilization method is promising for industrial applications. The method can also be expanded to other CDs and other hydrophobic compounds bioconversion.

The effect of 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes on the transformation of AD to 9α-OH-AD by Rhodococcus rhodochrous DSM43269.

Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9α-hydroxylases. However, the function of its 3-ketosteroid-Δ(1)-dehydrogenases (KSDD) remains unknown. This study compared the involvement of ksdds in the strain's androst-4-ene-3,17-dione (AD) transformation via gene deletion. The conversion was performed using AD as substrate or directly with 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD). The single deletion of ksdd1 or ksdd3 did not appear to result in the accumulation of 9α-OH-AD, whereas the single mutant △ksdd2 could preserve this compound to some extent. To further compare the role of ksdds in this strain, double mutants were constructed. All ksdd2 mutants combined with ksdd1 and/or ksdd3 resulted in the accumulation of 9α-OH-AD, among which the double mutant △ksdd2,3 behaved similarly to the single mutant △ksdd2 in this process. The mutant that lacked both ksdd1 and ksdd3 was still displayed, with no effect on the degradation of 9α-OH-AD. The triple mutant △ksdd1,2,3 was then constructed and exhibited the same capability as △ksdd1,2, accumulating more 9α-OH-AD than △ksdd2,3 and △ksdd2. The transcription of KSDD1 and KSDD2 increased, whereas that of KSDD3 seemed to exhibit no change, despite the use of the inducer AD or 9α-OH-AD. Thus, only ksdd1 and ksdd2 were involved in the transformation of AD to 9α-OH-AD. ksdd2 had the main role, ksdd1 had a minor effect on 9α-OH-AD degradation, and ksdd3 did not exhibit any action in this course.

Hydroxypropyl-ß-cyclodextrin-mediated alterations in cell permeability, lipid and protein profiles of steroid-transforming Arthrobacter simplex.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) enhances steroid 1-dehydrogenation biotransformation by Arthrobacter simplex. In this work, HP-ß-CD-induced improvement of A. simplex CPCC 140451 cell envelope permeability which had positive effects on the steroid bioconversion was confirmed by a comparative investigation which showed a lower dehydrogenase activity and higher cell permeability of the cells after being incubated with HP-ß-CD. Atomic force microscopy and transmission electron microscopy micrographs showed that HP-ß-CD altered the size, sharpness, and surface structure of the cell envelope. The analysis of lipid composition revealed that the proportion of extractable lipids decreased and the fatty acids profile was considerably altered. The contents of unsaturated fatty acids and long-chain fatty acids were reduced by 11.77 and 14.98%, respectively. The total leakage of protein level increased to 8%. Proteins belonging to the ATP-binding cassette superfamily and major facilitator superfamily were observed outside the cell. These alterations can explain the change of permeability on the molecular level under HP-ß-CD treatment. Results showed the material basis and mechanisms underlying the cellular changes, thus most likely contributing to the conversion rate in addition to cyclodextrins known effects on substrate solubility.

Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum.

Mycobacterium neoaurum TCCC 11028 (MNR) and M. neoaurum TCCC 11028 M3 (MNR M3) significantly differ in the ratio of androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) produced. The large fluctuations are related to the dehydrogenation activity of 3-ketosteroid-Δ(1)-dehydrogenase (KsdD). Analysis of the primary structure of KsdD showed that the Ser-138 of KsdD-MNR changed to Leu-138 of KsdD-MNR M3 because of C413T in the ksdD gene. This phenomenon directly affected KsdD activity. The effect of the primary structure of KsdD on dehydrogenation activity was confirmed through exogenous expression. Whole-cell transformation initially revealed that KsdD-MNR showed a higher dehydrogenation activity than KsdD-MNR M3. Then, ksdD gene replacement strain was constructed by homologous recombination. The results of steroid transformation experiments showed that the ability of the MNR M3ΔksdD::ksdD-MNR strain to produce ADD was improved and it returned to the similar level of the MNR strain. This result indicated that the ADD/AD ratio of the two M. neoaurum strains was influenced by the difference in ksdD. The mechanism by which residue mutations alter enzyme activity may be connected with the crystal structure of KsdD from Rhodococcus erythropolis SQ1. As a key amino acid residue in the active center position, Ser-138 played an important role in maintaining the active center in the hydrophobic environment of KsdD. This study may serve as a basis for future studies on the structural analysis and catalytic mechanism of dehydrogenase.

The effect of ethanol on cell properties and steroid 1-en-dehydrogenation biotransformation of Arthrobacter simplex.

Resting cells of Arthrobacter simplex with 1-en-dehydrogenation ability were prepared and treated by ethanol at subinhibitory concentrations (4%-15%, v/v), then added into the ethanol-free system containing low concentration of cortisone acetate (1 g L(-1)) to produce prednisone acetate by C1,2 dehydrogenation reaction. Results showed that, within the range of ethanol concentration, the initial conversion rate was varied significantly with the concentration of ethanol and the maximum was obtained at 8% (v/v) ethanol, which was increased by 32.6% compared with the control. A series of cell features closely relevant to biotransformation efficiency were further analyzed. It indicated that ethanol acting on cell wall and membrane could be used as a mediator to enhance cell permeability, which facilitated the penetration of substrate across cell barrier within a short time, resulting in the elevated initial conversation rate. The observation of fatty acids composition suggested that the increased unsaturated fatty acids, especially cis-isomers, in the presence of ethanol led to the disorganization of the native arrangement of lipids and thus increased cell permeability. Our findings demonstrated that another facilitation of ethanol was to promote substrate transport into cells by permeabilization, which would provide the guidance in the practical application of organic solvents in steroid biotransformation.