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Due to the diverse structures and broad functions of terpenes, microbial biosynthesis of these compounds has been favored as a sustainable alternative to phytoextraction and chemosynthesis. Here, systematic metabolic engineering strategies were explored in the oleaginous yeast Yarrowia lipolytica for hyperproducing sesquiterpene germacrene A which serves as an important intermediate of numerous anticarcinogens. By identifying the most efficient germacrene A synthase to date, reconstructing the endogenous mevalonate pathway and extending the cytosolic acetyl-CoA pool by regulating lipid metabolism, the resulting strain overproduced 2.794 g L−1 germacrene A in shake flasks, which represented a 38-fold improvement over the initial strain. The engineered strain was subsequently capable of producing 39 g L−1 germacrene A at a yield of 0.181 g g−1 glucose during optimized bioreactor fermentation, with this being the highest sesquiterpene production level reported to date for Y. lipolytica. These results demonstrate that reprogramming the metabolism of the host cell by systematic metabolic engineering plays an essential role in diverting its inherent metabolic fluxes for sesquiterpene biosynthesis and these approaches can be extensively applied for synthesizing natural terpenes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In steroid biotransformation, soybean oil can improve the productivity of steroids by increasing substrate solubility and strengthen the cell membrane permeability. However, little is known of its role as oxygen carrier and its mechanism of promoting the steroid biotransformation. In this work, soybean oil used as oxygen vector for the enhancement of androst-4-ene-3,17-dione (AD) production by Mycobacterium neoaurum TCCC 11979 ( MNR) was investigated. Upon the addition of 16% ( v/ v) soybean oil, the volumetric oxygen transfer coefficient ( K a) value increased by 44%, and the peak molar yield of AD (55.76%) was achieved. Analysis of intracellular cofactor levels showed high NAD, ATP level, and a low NADH/NAD ratio. Meanwhile, the two key enzymes of the tricarboxylic acid (TCA) cycle, namely, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, were upregulated after incubation with soybean oil. These enhancements induced by the increasing of oxygen supply showed positive effects on phytosterol (PS) bioconversion. Results could contribute to the understanding of effects of soybean oil as oxygen vector on steroid biotransformation and provided a convenient method for enhancing the efficiency of aerobic steroid biocatalysis. [ABSTRACT FROM AUTHOR]

Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9α-hydroxylases. However, the function of its 3-ketosteroid-Δ-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. [ABSTRACT FROM AUTHOR]

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-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 M3Δ ksdD:: 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. [ABSTRACT FROM AUTHOR]