Improving Biotransformation Efficiency of Arthrobacter simplex by Enhancement of Cell Stress Tolerance and Enzyme Activity.

Arthrobacter simplex exhibits excellent Δ1-dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.

Identification, Biological Characteristics, and Active Site Residues of 3-Ketosteroid Δ1-Dehydrogenase Homologues from Arthrobacter simplex.

3-Ketosteroid Δ1-dehydrogenase (KsdD) is the key enzyme responsible for Δ1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.

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.

IrrE Improves Organic Solvent Tolerance and Δ1-Dehydrogenation Productivity of Arthrobacter simplex.

During steroid bioconversion, organic solvents are widely used for facilitating hydrophobic substrate dissolution in industry. Thus, strains that tolerate organic solvents are highly desirable. IrrE, a global transcriptional factor, was introduced into Arthrobacter simplex with Δ1-dehydrogenation ability. The results evidenced that IrrE did not affect cell biological traits and biotransformation performance under non-stress conditions. However, the recombinant strain achieved a productivity higher than that of the control strain in systems containing more ethanol and substrate, which coincided with cell viability under ethanol stress, the major stress factor during biotransformation. It also demonstrated that IrrE caused genome-wide transcriptional perturbation, and several defense proteins or systems were linked with higher organic solvent tolerance. IrrE simultaneously enhanced cell resistance to various stresses, and its horizontal impacts showed strain and stress dependence. In conclusion, the introduction of exogenous global regulators is an efficient approach to enhance organic solvent tolerance in steroid-transforming strains, resulting in higher productivity.

1ß,15α-Dihy-droxy-16α,17-ep-oxy-pregn-4-ene-3,20-dione.

The title mol-ecule, C21H28O5, is composed of three six-membered rings (A/B/C) and a five-membered ring (D). Ring A adopts a 1α-sofa conformation, while rings B and C adopt chair conformations. Cyclo-pentane ring D adopts a 14α-envelope conformation. In the crystal, O-H⋯O hydrogen bonds lead to the formation of ribbons running along the a axis. The structure is further consolidated by C-H⋯O inter-actions, which link the molecules head-to-tail into ribbons along the a axis.

Influence of hydroxypropyl-ß-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum.

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-ß-cyclodextrin (HP-ß-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-ß-CD, conversion of 0.5 g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9 % compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-ß-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ¹-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-ß-CD.

Genome shuffling of Streptomyces gilvosporeus for improving natamycin production.

Improvement of natamycin production by Streptomyces gilvosporeus ATCC 13326 was performed by recursive protoplast fusion in a genome-shuffling format. After four rounds of genome shuffling, the best producer, GS 4-21, with genetic stability was obtained and its production of natamycin reached 4.69 ± 0.05 g/L in shaking flask after 96 h cultivation, which was increased by 97.1% and 379% in comparison with the highest parental strain pop-72A(r)07 and the initial strain ATCC 13326, respectively. Compared with the initial strain ATCC 13326, the recombinant GS 4-21 presented higher polymorphism. Fifty-four proteins showed differential expression levels between the recombinant GS 4-21 and initial strain ATCC 13326. Of these proteins, 34 proteins were upregulated and 20 proteins were downregulated. Of the upregulated proteins, one protein, glucokinase regulatory protein, was involved in natamycin biosynthesis. This comprehensive analysis would provide useful information for understanding the natamycin metabolic pathway in S. gilvosporeus.

11α,15α-Dihy-droxy-androst-4-ene-3,17-dione.

The title compound, C(19)H(26)O(4), was biotransformed from androstenedione. In the crystal, inter-molecular O-H⋯O hydrogen bonds link molecules into a corrugated sheet, which lies parallel to the ab plane. Ring A has a slightly distorted half-chair conformation, rings B and C adopt chair conformations, while the cyclo-pentane ring D adopts a 14α-envelope conformation.