An Escherichia coli Nissle 1917-based live therapeutics platform with integrated phage resistance and programmable temperature sensitivity.

Live bacterial therapeutics (LBT) represent a transformative modality for managing refractory chronic diseases. However, the absence of optimized microbial chassis systems is a significant barrier to clinical translation. To bridge this gap, we engineered Escherichia coli Nissle 1917 (EcN) into a versatile platform that meets the requirements for strain development and clinical application.

Metabolic Engineering of Acinetobacter baylyi ADP1 for L-Leucine Production.

Acinetobacter baylyi ADP1 has garnered attention as a promising synthetic biology chassis due to its compact genome, rapid growth, innate competence for horizontal gene transfer, and ease of genetic manipulation. To assess its potential for natural product biosynthesis, we engineered ADP1 for the production of l-leucine. First, feedback inhibition was relieved by overexpressing the endogenous leuA and ilvBN genes, alongside the replacement of transcriptional attenuation regions within the leuBCD operon.

Synergistic increase in coproporphyrin III biosynthesis by mitochondrial compartmentalization in engineered .

Coproporphyrin III (CP III), a natural porphyrin derivative, has extensive applications in the biomedical and material industries. has previously been engineered to highly accumulate the CP III precursor 5-aminolevulinic acid (ALA) through the C4 pathway. In this study, a combination of cytoplasmic metabolic engineering and mitochondrial compartmentalization was used to enhance CP III production in .

Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme.

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood.

Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum.

Establishment of an Efficient Expression and Regulation System in for Economical and High-Level Production of the Natural Blue Pigment Indigoidine.

Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from species. However, due to the complex metabolic switches of , most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of , here, an updated regulation system derived from the self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in TK24.

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons.

Background: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct.

Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes.

Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide.

Background: The conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process.

Improving the biotransformation efficiency of soybean phytosterols in by the combined deletion of and in cell envelope synthesis.

Biotransformation of soybean phytosterols into 9-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by , , and was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope.

Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum.

The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum.

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum.

Background: The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M.

Metabolic Adaptation of Mycobacterium neoaurum ATCC 25795 in the Catabolism of Sterols for Producing Important Steroid Intermediates.

To understand the adaptation of Mycobacterium neoaurum ATCC25795 ( Mn) in sterol catabolism and steroid production, we used integrated transcriptome and proteome analysis to identify the biochemical pathways utilized in this process. Metabolic alterations during sterol catabolism center on propionyl-CoA pools. Generally, enhanced pathways for metabolizing propionyl-CoA were found in Mn, which were tightly coordinated with cell-envelope biosynthesis.

Integrated Transcriptome and Proteome Studies Reveal the Underlying Mechanisms for Sterol Catabolism and Steroid Production in Mycobacterium neoaurum.

Integrated transcriptome and proteome studies were performed to investigate sterol biotransformation in wild-type Mycobacterium neoaurum ATCC 25795 ( Mn) and the mutant strains producing steroid intermediates. Transcriptome and proteome studies indicated that several metabolic activities were noticeably dynamic, including cholesterol degradation, central carbon metabolism, cell envelope biosynthesis, glycerol metabolism, and transport. Interestingly, a poor overall correlation between mRNA and translation profiles was found, which might contribute to the metabolic adaptation in cholesterol catabolism.

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1 and KshA2, were characterized and further engineered in a sterol-digesting strain, ATCC 25795, to construct androstenone-producing strains.

Enhancement of 9α-Hydroxy-4-androstene-3,17-dione Production from Soybean Phytosterols by Deficiency of a Regulated Intramembrane Proteolysis Metalloprotease in Mycobacterium neoaurum.

Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes.

Background: The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols.

Role Identification and Application of SigD in the Transformation of Soybean Phytosterol to 9α-Hydroxy-4-androstene-3,17-dione in Mycobacterium neoaurum.

9α-Hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid pharmaceutical intermediate which can be produced by the conversion of soybean phytosterols in mycobacteria. However, the unsatisfactory productivity and conversion efficiency of engineered mycobacterial strains hinder their industrial applications. Here, a sigma factor D (sigD) was investigated due to its dramatic downregulation during the conversion of phytosterols to 9-OHAD.