Metabolic engineering of Escherichia coli for microbial synthesis of monolignols.

Monolignols are important plant metabolites involved in lignin biosynthesis. Their derivatives exhibit various physiological and pharmaceutical functions. Here, efficient enzymes were selected to construct p-coumaryl alcohol biosynthetic pathway and the titer reached 501.8±41.4mg/L under optimized conditions. The pathway was further extended to produce caffeyl alcohol and coniferyl alcohol by introducing a hydroxylase and methyltransferases. However, the promiscuity of the hydroxylase HpaBC led to the formation of an instable intermediate L-dopa from tyrosine, causing loss of the carbon sources. To solve this problem, microbial co-cultures were designed to minimize the accessibility of HpaBC to tyrosine. With the optimal inoculation ratio, 401.0±15.3mg/L of caffeyl alcohol was produced, which is nearly 12 times higher than that of the mono-culture. The titer reached 854.1±44.6mg/L in scale-up production. The same strategy was used for coniferyl alcohol production. Limited by the activity of methyltransferases, the highest titer was 124.9±5.1mg/L with 232.9±15.1mg/L of caffeyl alcohol accumulated. To the best of our knowledge, this is the first report about microbial production of caffeyl alcohol and coniferyl alcohol. This work also demonstrated the promising potential of microbial co-cultures for prevention of side-reactions.

[1]  Gregory Stephanopoulos,et al.  Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli. , 2016, Biotechnology journal.

[2]  James C Liao,et al.  Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass , 2013, Proceedings of the National Academy of Sciences.

[3]  M. Ikeda.  Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering , 2006, Applied Microbiology and Biotechnology.

[4]  C. Collins,et al.  Modular optimization of multi-gene pathways for fatty acids production in E. coli , 2013, Nature Communications.

[5]  A. Burgard,et al.  Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. , 2011, Nature chemical biology.

[6]  J. Liao,et al.  Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels , 2008, Nature.

[7]  A. Fiorentino,et al.  Isolation, characterization, and antioxidant activity of E- and Z-p-coumaryl fatty acid esters from cv. Annurca apple fruits. , 2005, Journal of agricultural and food chemistry.

[8]  Kechun Zhang,et al.  Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine , 2010, Proceedings of the National Academy of Sciences.

[9]  Zhe-zhi Wang,et al.  Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation. , 2014, Metabolic engineering.

[10]  Mattheos A G Koffas,et al.  Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. , 2016, Metabolic engineering.

[11]  J. Nam,et al.  Anti‐inflammatory activity of p‐coumaryl alcohol‐γ‐O‐methyl ether is mediated through modulation of interferon‐γ production in Th cells , 2009, British journal of pharmacology.

[12]  M. R. Hemm,et al.  New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Stephanopoulos,et al.  Engineering Escherichia coli coculture systems for the production of biochemical products , 2015, Proceedings of the National Academy of Sciences.

[14]  Patrik R. Jones,et al.  Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities , 2012, Proceedings of the National Academy of Sciences.

[15]  Yajun Yan,et al.  Caffeic acid production enhancement by engineering a phenylalanine over‐producing Escherichia coli strain , 2013, Biotechnology and bioengineering.

[16]  Y. Chao,et al.  Potential production platform of n-butanol in Escherichia coli. , 2015, Metabolic engineering.

[17]  Haoran Zhang,et al.  Modular co-culture engineering, a new approach for metabolic engineering. , 2016, Metabolic engineering.

[18]  Mark A Eiteman,et al.  Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains , 2012, Microbial Cell Factories.

[19]  J. Nielsen,et al.  De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae. , 2015, Metabolic engineering.

[20]  Luke A. Gilbert,et al.  Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds , 2015, Cell.

[21]  G. Stephanopoulos,et al.  Improving fatty acids production by engineering dynamic pathway regulation and metabolic control , 2014, Proceedings of the National Academy of Sciences.

[22]  C. Wittmann,et al.  From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. , 2011, Metabolic engineering.

[23]  V. Křen,et al.  Silybin and silymarin--new and emerging applications in medicine. , 2007, Current medicinal chemistry.

[24]  Frank Jansen,et al.  Metabolic engineering for p‐coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway , 2014, Biotechnology and applied biochemistry.

[25]  Qipeng Yuan,et al.  Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli. , 2014, Metabolic engineering.

[26]  Qipeng Yuan,et al.  Combinatorial biosynthesis of plant-specific coumarins in bacteria. , 2013, Metabolic engineering.

[27]  J. Ralph,et al.  Facile large-scale synthesis of coniferyl, sinapyl, and p-coumaryl alcohol , 1992 .

[28]  Qipeng Yuan,et al.  Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin , 2013, Nature Communications.

[29]  C. Chapple,et al.  Rewriting the lignin roadmap. , 2002, Current opinion in plant biology.

[30]  T. Hanai,et al.  Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch. , 2014, Metabolic engineering.

[31]  D. Oancea,et al.  Peroxidase-mediated oxidation of l-dopa: A kinetic approach , 2010 .

[32]  Yajun Yan,et al.  Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex , 2012, Microbial Cell Factories.

[33]  J. Liao,et al.  Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping. , 2010, Journal of bioscience and bioengineering.

[34]  J. Keasling,et al.  High-level semi-synthetic production of the potent antimalarial artemisinin , 2013, Nature.

[35]  James C. Liao,et al.  Expanding metabolism for biosynthesis of nonnatural alcohols , 2008, Proceedings of the National Academy of Sciences.

[36]  G. Stephanopoulos,et al.  Distributing a metabolic pathway among a microbial consortium enhances production of natural products , 2015, Nature Biotechnology.