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Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co‐utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose‐to‐glucose flux ratio. A mutant copy of the xylose‐specific activator (XylR) was then introduced into the phenylalanine‐overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose−xylose co‐utilization. Carbon contribution analysis through 13C‐fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose−xylose mixture; a threefold increase over NST74. Then, using biomass‐derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9‐fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L‐h). This study presents a novel strategy for enhancing phenylalanine production through the co‐utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products. [ABSTRACT FROM AUTHOR]

Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with a wide range of industrial and commercial applications. Significant progress has been made to alternatively and sustainably produce many aromatics from renewable substrates using microbial biocatalysts. While the construction of both natural and non‐natural pathways has expanded the number and diversity of aromatic bioproducts, pathway modularization in both single‐ and multi‐strain systems continues to support the enhancement of key production metrics towards economically‐viable levels. Emerging tools for implementing more precise metabolic control (e.g. CRISPRi, sRNA) as well as the engineering of novel high‐throughput screening platforms utilizing in vivo aromatic biosensors, meanwhile, continue to facilitate further optimization of both pathways and hosts. While product toxicity persists as a key challenge limiting the production of many aromatics, various successful strategies have been demonstrated towards improving tolerance, including via membrane and efflux pump engineering as well as by exploiting alternative production hosts. Finally, as a further step towards sustainable and economical aromatic bioproduction, non‐model substrates including lignin‐derived compounds continue to emerge as viable feedstocks. This review highlights recent and notable achievements related to such efforts while offering future outlooks towards engineering microbial cell factories for aromatic production. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

ABSTRACT The non-renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non-natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate p-hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL-dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745-1754. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]