Background:The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C 5 , C 6 , and C 12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis. Results:Enabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR-Cas9 system from Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde. Conclusions:Newly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPRCas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1-7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate.

Fermentation of lignocellulosic sugar mixtures is often suboptimal due to inefficient xylose catabolism and sequential sugar utilization caused by carbon catabolite repression. Unlike in conventional applications employing a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting the unique community features, including modularity, division of labor, and facile tunability. A series of synthetic, catabolically orthogonal coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W or ethanologenic LY180. Net catabolic activities were effectively balanced by simple tuning of the inoculum ratio between specialist strains, which enabled coutilization (98% of 100 g L −1 total sugars) of glucose−xylose mixtures (2:1 by mass) for both culture systems in simple batch fermentations. The engineered ethanologenic cocultures achieved ethanol titer (46 g L −1 ), productivity (488 mg L −1 h −1 ), and yield (∼90% of theoretical maximum), which were all significantly increased compared to LY180 monocultures.

Flores et al. Lactate and Succinate Production by Co-cultures simple batch fermentation. High product titer (88 g L −1 D-lactate, 84 g L −1 succinate) and maximum productivity (2.5 g L −1 h −1 D-lactate, 1.3 g L −1 h −1 succinate) and product yield (0.97 g g-total sugar −1 for D-lactate, 0.95 g g-total sugar −1 for succinate) were also achieved.

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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