A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach.

Dynamic flux regulation for high-titer anthranilate production by plasmid-free, conditionally-auxotrophic strains of Pseudomonas putida.

Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii.

Spatiotemporal Manipulation of the Mismatch Repair System of Accelerates Phenotype Emergence.

The development of complex phenotypes in industrially relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed toward increasing mutation frequencies, yet the precise control of mutagenesis processes in cell factories continues to represent a significant technical challenge. species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in the bacterial domain.

Evolutionary Approaches for Engineering Industrially Relevant Phenotypes in Bacterial Cell Factories.

The bio-based production of added-value compounds (with applications as pharmaceuticals, biofuels, food ingredients, and building blocks) using bacterial platforms is a well-established industrial activity. The design and construction of microbial cell factories (MCFs) with robust and stable industrially relevant phenotypes, however, remains one of the biggest challenges of contemporary biotechnology. In this review, traditional and cutting-edge approaches for optimizing the performance of MCFs for industrial bioprocesses, rooted on the engineering principle of natural evolution (i.

Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida.

The core metabolism for glucose assimilation of the soil bacterium and platform strain Pseudomonas putida KT2440 has been reshaped from the native, cyclically-operating Entner-Doudoroff (ED) pathway to a linear Embden-Meyerhof-Parnas (EMP) glycolysis. The genetic strategy deployed to obtain a suitable host for the synthetic EMP route involved not only eliminating enzymatic activities of the ED pathway, but also erasing peripheral reactions for glucose oxidation that divert carbon skeletons into the formation of organic acids in the periplasm. Heterologous glycolytic enzymes, recruited from Escherichia coli, were genetically knocked-in in the mutant strain to fill the metabolic gaps for the complete metabolism of glucose to pyruvate through a synthetic EMP route.

New Insights on Steroid Biotechnology.

Nowadays steroid manufacturing occupies a prominent place in the pharmaceutical industry with an annual global market over $10 billion. The synthesis of steroidal active pharmaceutical ingredients (APIs) such as sex hormones (estrogens, androgens, and progestogens) and corticosteroids is currently performed by a combination of microbiological and chemical processes. Several mycobacterial strains capable of naturally metabolizing sterols (e.

Unravelling a new catabolic pathway of C-19 steroids in Mycobacterium smegmatis.

In this work, we have characterized the C-19+ gene cluster (MSMEG_2851 to MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis, we have identified the genes encoding enzymes involved in the modification of the A/B steroid rings during the catabolism of C-19 steroids in certain M. smegmatis mutants mapped in the PadR-like regulator (MSMEG_2868), that constitutively express the C-19+ gene cluster.

Bioconversion of Phytosterols into Androstadienedione by Mycobacterium smegmatis CECT 8331.

The C19 steroid 1,4-androstadiene-3,17-dione (androstadienedione, ADD) is an added value product used as a synthon in the pharmaceutical industry for the commercial production of corticosteroids, mineralocorticoids, oral contraceptives, and other pharmaceutical steroids. Phytosterol biotransformation catalyzed by microbial whole cells is actually a very well-established research area in white biotechnology. The protocol below provides detailed information on ADD production by the mutant CECT 8331 of Mycobacterium smegmatis mc155 using phytosterols as raw material in a lab scale.

Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis.

The C-19 steroids 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) or 9α-hydroxy-4-androstene-3,17-dione (9OH-AD), which have been postulated as intermediates of the cholesterol catabolic pathway in Mycobacterium smegmatis, cannot be used as sole carbon and energy sources by this bacterium. Only the ΔkstR mutant which constitutively expresses the genes repressed by the KstR regulator can metabolize AD and ADD with severe difficulties but still cannot metabolize 9OH-AD, suggesting that these compounds are not true intermediates but side products of the cholesterol pathway. However, we have found that some M.

Engineering Mycobacterium smegmatis for testosterone production.

A new biotechnological process for the production of testosterone (TS) has been developed to turn the model strain Mycobacterium smegmatis suitable for TS production to compete with the current chemical synthesis procedures. We have cloned and overexpressed two genes encoding microbial 17β-hydroxysteroid: NADP 17-oxidoreductase, from the bacterium Comamonas testosteroni and from the fungus Cochliobolus lunatus. The host strains were M.

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons.

A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multi-step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains.