Artificial enzyme cascades are constructed by the combination of biocatalysts that are metabolically unrelated in nature. Rapidly increasing numbers of available biocatalysts and cross-disciplinary efforts such as biocatalytic retrosynthesis accelerated the design of (bio)synthetic routes with increasing complexity to produce value-added chemicals. Well-established genetic regulatory elements (e.g., promoters) for balancing enzyme production, complementing substrate channeling approaches, and the engineering of enzymatic host backgrounds by gene knock-out target different molecular levels to implement and optimize pathways in whole cells. The introduction of nonnative enzymes may interfere with the metabolic environment in hosts like Escherichia coli (E. coli), which might impair the carbon flux through the synthetic pathway in vivo. ^Particularly, unexpected interactions between different synthetic genetic elements are often underestimated contextual issues in pathway design and a current challenge. Consequently, this thesis aims at the application of synthetic enzyme cascades in vivo and the resolution of both compositional and host context dependencies by complementing flux enhancement strategies to maximize product titers. The biocatalytic retrosynthetic approach pursued in this thesis, revealed two distinct pathway designs to produce polyhydroxylated compounds. Both involve the oxidation of primary alcohols to the corresponding aldehydes and subsequent carboligation catalyzed by an aldolase. Regarding the second cascade step, aldols can be either produced from extracellularly added aldol donor molecules such as (di)hydroxyacetone [(D)HA] or by hijacking glycolytic DHA phosphate (DHAP). The latter will tightly interconnect the de novo pathway and the central carbon metabolism of E. ^coli and circumvent the lability of DHAP in vitro. The implementation of a phosphatase and stereocomplementary DHAP-dependent aldolases dephosphorylates the phosphorylated intermediate adduct to shift the reaction equilibrium and provides access to aldol products in different configurations, respectively. Subsequent screening of the biocatalytic toolbox identified AlkJ, an alcohol dehydrogenase from Pseudomonas putida, as an efficient biocatalyst for the in situ production of reactive aldehyde acceptors. For the first cascade design, the (D)HA-dependent aldolase Fsa1-A129S from E. coli was most suitable yielding aldol adducts with (3S,4R) configuration, whereas, for the second pathway, the DHAP-dependent FucA (E. coli) was selected to produce (3R,4R) polyhydroxylated compounds and phosphatases from different microbial hosts studied. Up-to-date sequence- and ligation-independent cloning techniques were successfully applied to assemble pathway modules in different genetic architectures. Compositional context was improved by the integration of multiple genetic (transcriptional) regulators including (synthetic) terminators to balance pathway enzyme production in vivo. The cellular host environment severely interfered with the in situ preparation of (cytotoxic) aldehyde intermediates by the rapid reduction to the alcoholic substrates and the irreversible metabolization to the corresponding carboxylic acids. These context issues were addressed by the utilization of highly engineered strains such as E. coli RARE and simply by the introduction of a reversing enzymatic activity, respectively, to reroute the carbon flux from the carboxylate sink toward cascade aldehyde intermediates. The latter employed a carboxylic acid reductase from Nocardia iowensis (CARNi). In the presence of AlkJ and CARNi, reactive aldehyde species were quickly interconverted between the corresponding alcohols and carboxylates. Consequently, aldehydes were equilibrated below nonviable, yet freely available concentrations for subsequent aldol reaction. This so far neglected strategy establishing a ‘hidden reservoir for reactive aldehyde species increased cell viability and could address the issue of aldehyde toxicity and persistence in vivo. Although aldols could not be synthesized via the DHAP-dependent cascade, crucial bottlenecks such as insufficient intracellular DHAP concentrations were identified by metabolomic analysis. To compensate this ‘parasitic interaction, a DHA kinase from Citrobacter freundii was successfully studied in this thesis and offers an optimization strategy for future applications. The research conducted in this thesis not only designed, assembled, implemented, and optimized an artificial biosynthetic pathway consisting of up to three metabolically unrelated enzymes (AlkJ, CARNi, Fsa1-A129S). The ‘hidden aldehyde reservoir approach in combination with a refined solid phase extraction purification, tackling the issue of notoriously low yielding aldol reactions in vitro, demonstrated the applicability of the developed system and synthesized structurally different aldols from the donor molecules HA and DHA in up to 91% isolated yields in short reaction times in living cells.