Light-driven progesterone production by InP–(M. neoaurum) biohybrid system

Abstract

Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic–biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum. Graphical Abstract

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

“…The construction of plasmids with functional monooxygenase systems for co-expression of proteins in bacterial cells has been described in several papers [ 14 , 16 , 19 , 21 ]. For co-expression of the protein components of the CH/L system, several polycistronic plasmids based on pBar and pTrc99A were constructed [ 14 , 21 ].…”

“…Mycolicibacterium neoaurum ). The combination of the recombinant M. neoaurum cells and InP nanoparticles for light regeneration of NADPH provided an increase in progesterone synthesis up to 235 ± 50 mg/L [ 19 ].…”

Engineering Electron Transfer Pathway of Cytochrome P450s

“…Lee et al [ 95 ] used eosin Y as a photosensitive dye, TEOA as an electron donor, and [Cp*Rh (bpy) H 2 O] as an electron medium to drive NADPH regeneration and achieve O-dealkylation catalyzed by P450 BM3 variants (Y51F/F87A, BM3M2). Liu et al [ 142 ] regenerated NADPH by combining engineered Bacillus auriculare with InP nanoparticles, which increased NADPH levels by 84 times.…”

Revisiting Solar Energy Flow in Nanomaterial-Microorganism Hybrid Systems

“…Following similar procedures, InP nanoparticles were prepared and hybridized with M. neoaurum to enhance progesterone production, and with N. sphaeroides to augment photosynthetic ethylene production, respectively. 149,152 However, the E CB of InP (−0.43 V vs NHE) makes it unlikely to catalyze the reduction of several important intracellular electron carriers, such as the ferredoxin (standard redox potential ranging between −0.4 V and −0.5 V vs NHE) or the methyl viologen (MV), a commonly used synthetic electron shuttle with a standard one-electron redox potential of −0.46 V vs NHE. Through strategies like doping and heterojunctions, the energy capture properties of the resulting materials could be fine-tuned.…”

“…This often necessitates additional surface modification or chemical bonding methods to stabilize material-cell interface, which can indirectly impact the quality of energy transfer. 94,132,149,192 Typically, E. coli cells were modified with cationic poly (allylamine hydrochloride) (PAH) to convert their negative surface potential to positive, allowing assembly with negatively charged I-HTCC nanomaterial. 133 InP nanoparticles coated with negatively charged tannic acid(TA)-Fe 3+ coordination networks were attached to the surface of engineered S. cerevisiae cells, which had been altered with PAH to acquire positive charges.…”