Feng-Qing Wang

An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells.

To reduce the interference in MTT reduction assay, we developed an improved protocol for evaluating the viability of Escherichia coli cells by conducting MTT reduction in 1.5-mL centrifuge tubes and formazan dissolution in test tubes. Our study is helpful in developing protocols for measuring the viability of other gram-negative bacteria.

Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols

3-Ketosteroid-△(1)-dehydrogenase (KstD) is a key enzyme involved in the microbial catabolism of sterols. Here, three homologues of KstD were characterized from Mycobacterium neoaurum ATCC 25795, showing distinct substrate preferences and transcriptional responses to steroids. Single deletion of any MN-kstD failed to result in a stable and maximum accumulation of 9-OHAD due to residual KstD activities. To develop stable 9-OHAD producers, all of these MN-KstDs were inactivated, which led to about 6.02g l(-1) of 9-OHAD from 15g l(-1) of phytosterols. However, the product was mixed with 1.55g l(-1) of AD as a major by-product. To transform AD, the oxygenase component of 3-ketosteroid-9α-hydroxylase (KSH), encoded by kshA, was overexpressed. As a result, the yield of 9-OHAD increased to 7.33g l(-1) with less than 0.31g l(-1) of AD and the selectivity of 9-OHAD production was improved to 95-97% among metabolites.

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87g/l androst-1,4-diene-3,17-dione and 4.53g/l androst-4-ene-3,17-dione, respectively.

Ammonia-containing dimethyl sulfoxide: an improved solvent for the dissolution of formazan crystals in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

To reduce interference with the dissolution of formazan crystals in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, we conducted a systematic investigation to study the effects of various buffers, HCl, NaOH, and ammonia. As a result, we identified an improved solvent, alkaline dimethyl sulfoxide (DMSO) containing 8 to 800 mM ammonia, which could dissolve formazan crystals in approximately 10 min so as to give a stable spectrum by eliminating buffering effects of the residual medium.

A comparative study of β-1, 4-endoglucanase (possessing β-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3).

β-1, 4-Endoglucanase (EG) from Bacillus subtilis LH was expressed in Escherichia coli Rosetta (DE3) and Pichia pastoris GS115, respectively. The CMCase activity of EG (EGE) from the cell lysate of DE3 reached 20,010U/ml, and that of EG (EGP) from the supernatant of GS115 was only 2008U/ml. EGE and EGP were bifunctional cellulases excluding β-1, 4-glucosidase (BGL). The CMCases of them, optimally active at 65°C and pH 6.8, exhibited more than 80% residual activity at pH 5-10 and 60% activity at 40-70°C and pH 5-9. EGE (EGP) mixed with BGL had more than 1.5-fold higher CMCase and filter paperase activities compared to EGE (EGP). N-glycosylation protected EGP from immobilized-papain attack and accounted for 30kDa and a higher thermostability, whereas EGE was decomposed into a 33kDa active truncated EG (EGT) and two 18kDa fragments. EGE and EGP performed much better than EGT in denim biostoning.

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

Comparison of methods for measuring viable E. coli cells during cultivation: Great differences in the early and late exponential growth phases

Four methods, namely enumeration of colony-forming units (CFU), aerobic respiration, MTT reduction capacity and succinate dehydrogenase activity were compared to determine the viability of E. coli cells at the early and late exponential growth phases. Our results revealed that great differences in cell viability existed between these methods and that the choice of method to determine cell viability must be made with caution.

Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in Streptomyces virginiae IBL-14

ABSTRACT A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.

Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris.

Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein-protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields.