Engineering Pseudomonas putida KT2440 for the production of isobutanol.

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback-resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions.

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum.

BACKGROUND: l-Histidine biosynthesis is embedded in an intertwined metabolic network which renders microbial overproduction of this amino acid challenging. This is reflected in the few available examples of histidine producers in literature. Since knowledge about the metabolic interplay is limited, we systematically perturbed the metabolism of Corynebacterium glutamicum to gain a holistic understanding in the metabolic limitations for l-histidine production. We, therefore, constructed C. glutamicum strains in a modularized metabolic engineering approach and analyzed them with LC/MS-QToF-based systems metabolic profiling (SMP) supported by flux balance analysis (FBA). RESULTS: The engineered strains produced l-histidine, equimolar amounts of glycine, and possessed heavily decreased intracellular adenylate concentrations, despite a stable adenylate energy charge. FBA identified regeneration of ATP from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as crucial step for l-histidine production and SMP identified strong intracellular accumulation of inosine monophosphate (IMP) in the engineered strains. Energy engineering readjusted the intracellular IMP and ATP levels to wild-type niveau and reinforced the intrinsic low ATP regeneration capacity to maintain a balanced energy state of the cell. SMP further indicated limitations in the C1 supply which was overcome by expression of the glycine cleavage system from C. jeikeium. Finally, we rerouted the carbon flux towards the oxidative pentose phosphate pathway thereby further increasing product yield to 0.093 ± 0.003 mol l-histidine per mol glucose. CONCLUSION: By applying the modularized metabolic engineering approach combined with SMP and FBA, we identified an intrinsically low ATP regeneration capacity, which prevents to maintain a balanced energy state of the cell in an l-histidine overproduction scenario and an insufficient supply of C1 units. To overcome these limitations, we provide a metabolic engineering strategy which constitutes a general approach to improve the production of ATP and/or C1 intensive products.

Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.