Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum.

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔkstD, that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A, MFKDΔhsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔhsd4AΔfadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17ß-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Functional analysis of acyl-CoA dehydrogenases and their application to C22 steroid production.

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression.

Actinoplanes aureus sp. nov., a novel protease-producing actinobacterium isolated from soil.

A novel protease-producing actinobacterium, designated strain NEAU-A11T, was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China, and characterised using a polyphasic approach. The hydrolytic enzymes, such as proteases, played critical roles in destruction of fungi by degrading the protein linkages to disrupt integrity in the cell wall. This suggested that the isolate could be a good biocontrol candidate against pathogens to control fungal diseases. On the basis of 16S rRNA gene sequence analysis, strain NEAU-A11T was indicated to belong to the genus Actinoplanes and was most closely related to Actinoplanes rectilineatus JCM 3194 T (98.9%). Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were arabinose, xylose and glucose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major fatty acids were C18:0, C16:0, C18:1 ω9c, C17:0 and C15:0. Genome sequencing revealed a genome size of 10,742,096 bp, a G + C content of 70.5% and 9,514 protein-coding genes (CDS), including 102 genes coding for protease. Moreover, Genome analysis showed that strain NEAU-A11T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Genome mining analysis using antiSMASH 5.0 led to the identification of 20 putative gene clusters responsible for the production of diverse secondary metabolites. Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with A. rectilineatus JCM 3194 T in the genus Actinoplanes. Whole-genome phylogeny showed strain NEAU-A11T formed a stable phyletic line with Actinoplanes lutulentus DSM 45883 T (97.6%). However, whole-genome average nucleotide identity value between strain NEAU-A11T and its reference strains A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24.8% (22.5-27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95-96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G + C in genomic DNA was 70.5%, within the range of 67-76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11T (= CCTCC AA 2019063 T = JCM 33971 T) as the type strain.

A ras GTPase-activating protein-binding protein, TaG3BP, associated with the modulation of male fertility in a thermo-sensitive cytoplasmic male sterile wheat line.

Hybrid wheat development may contribute to higher, more stable yield and could result in greater food security for much of the world's growing population. YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat lines were developed for use in hybrid wheat breeding in China. To investigate the molecular mechanism of modulation of male fertility in the YS-type TCMS wheat lines, a ras GTPase-activating protein-binding protein (TaG3BP) was examined. The deduced amino acid sequence encoded by TaG3BP was conserved in the sequenced genomes of Embryophyte. TaG3BP expression in the anthers of YS-type TCMS lines taken at the critical fertility reversion stage of pollen development from male fertile anthers was higher than that from male-sterile anthers, either by quantitative real-time PCR or by western blot analysis. Sequence analysis on the cDNA and genomic DNA of TaG3BP in three kinds of K-type CMS wheat lines and their maintainers indicated that there were no significant difference between the genes or in their 5' flanking sequences. The TaG3BP expression revealed by quantitative real-time RT-PCR was lower in the young spikes of these CMS lines than that of their maintainers. This indicates that TaG3BP expression is associated with the modulation, from male-sterile to fertile, of the TCMS wheat line, and TaG3BP might be a key factor in the pathway responsible for the fertility reversion.

[Cloning and expression analysis of drought-tolerant and wa-ter-saving related gene PmMYB in broomcorn millet].

Exploring novel genes and gene combinations are of primary importance in plant drought-tolerance and water-saving research. Based on the EST sequence of MYB gene obtained in the research on molecular basis of drought-tolerance and water-saving in broomcorn millet (Panicum miliaceum L.), a 1 739 bp genomic sequence of MYB gene, designated PmMYB, was amplified using primers based on this EST sequence and that of rice MYB18. Sequence analysis indicated that PmMYB consisted of 2 introns, 121 bp (347-467 bp) and 93 bp (599-691 bp) in length respectively, and 3 extrons. The full-length cDNA sequence of PmMYB is 1 525 bp, which includes 212 bp 3' untranslated region, 41 bp 5' untranslated region, and 1 272 bp coding region which encodes 424 amino acids with a serine-rich region. PmMYB is a typical R2, R3-MYB transcription factor with its 2 typical DNA-binding domains (amino acid sites of 13-63 and 66-114). The multiple alignment of R2, R3 amino acid repeat regions of MYB genes among broomcorn millet, rice, corn, loblolly pine, Arabidopsis, capsicum, upland cotton, barley and aubergine indicates that R2, R3 repeat regions are very conserved. The phylogenetic analysis based on amino acid sequence shows that MYB genes are highly divergent with similarity ranging from 32% to 84%. PmMYB has the highest similarity (84%) with MYB18 gene of rice, whereas 46% and 41% with barley and corn, respectively. Semi-quantitative RT-PCR confirmed that PmMYB was upregulated by water stress and re-watering in broomcorn millet. Cloning this gene may provide a good foundation to improve the drought-tolerance and water-saving in broomcorn millet as well as in other plants.

[Representativeness of Northeast China spring soybeans and their genetic diversity at SSR loci].

A total of 283 accessions were selected from the total 3 226 Northeast China spring soybeans, which represented > 80% of the whole based on their qualitative and quantitative traits. The representative samples were analyzed by 61 SSR loci, and a total of 534 alleles were detected, ranging 2 - 16 alleles per locus, with an average of 8.75 alleles per locus. Among the accessions, the Simpson diversity index (SDI) for each locus ranged from 0.406 to 0.886, with a mean of 0.704, which was relatively lower since there were dominant alleles at most of loci in the representative samples. 35 accessions had specific alleles, which distributed among 29 loci. The differentiation coefficient was lower than 9.27% in 61 loci among the three provinces of Northeast China, which might be caused by lots of common alleles shared among these provinces. The genetic diversity in Jilin and Liaoning provinces was nearly equal, but was higher than that in Heilongjiang Province. The landraces of the representative samples at 61 loci had a higher genetic diversity than that of the released cultivars. The genetic diversity appeared within and between the provinces could be used to broaden the genetic base of modern cultivars, and also, the landraces were the major source for soybean breeding because of their high genetic diversity.