Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose.

In nature glucose is a common carbon and energy source for catabolic use and also a building unit of polysaccharides and glycosylated compounds. The presence of strong glucose catabolic pathways in microorganism rapidly decomposes glucose into smaller metabolites and challenges non-catabolic utilization of glucose as C6 building unit or precursor. To address this dilemma, we design a synergetic carbon utilization mechanism (SynCar), in which glucose catabolism is inactivated and a second carbon source (e.g. glycerol) is employed to maintain cell growth and rationally strengthen PEP driving force for glucose uptake and non-catabolic utilization. Remarkably, a trehalose biosynthesis model developed for proof-of-concept indicates that SynCar leads to 131% and 200% improvement in trehalose titer and yield, respectively. The conversion rate of glucose to trehalose reaches 91% of the theoretical maximum. This work demonstrates the broad applicability of SynCar in the biosynthesis of molecules derived from non-catabolic glucose.

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

[2]  M. Eiteman,et al.  Optimization of the ion-exchange analysis of organic acids from fermentation , 1997 .

[3]  J. Keasling,et al.  Microbial engineering for the production of advanced biofuels , 2012, Nature.

[4]  M. Follettie,et al.  Metabolic engineering of Corynebacterium glutamicum. , 1994, Annals of the New York Academy of Sciences.

[5]  T. Inada,et al.  Accumulation of Glucose 6-Phosphate or Fructose 6-Phosphate Is Responsible for Destabilization of Glucose Transporter mRNA inEscherichia coli * , 2003, The Journal of Biological Chemistry.

[6]  R. Linhardt,et al.  Production of chondroitin in metabolically engineered E. coli. , 2015, Metabolic engineering.

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

[8]  A. Goldberg,et al.  Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  B. Doublet,et al.  Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains. , 2008, Journal of microbiological methods.

[10]  Peng Xu,et al.  Pathway and protein engineering approaches to produce novel and commodity small molecules. , 2013, Current opinion in biotechnology.

[11]  Mark R Prausnitz,et al.  Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity. , 2010, Journal of controlled release : official journal of the Controlled Release Society.

[12]  Sung Kuk Lee,et al.  Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. , 2015, Metabolic engineering.

[13]  Jian Liu,et al.  Design of Biologically Active Heparan Sulfate and Heparin Using an Enzyme‐Based Approach , 2009 .

[14]  Alfredo Martínez,et al.  Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products , 2003, Biotechnology and bioengineering.

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

[16]  M. Koffas,et al.  Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. , 2016, Biotechnology advances.

[17]  Mattheos A. G. Koffas,et al.  Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli , 2005, Applied and Environmental Microbiology.

[18]  Duckhee Lee,et al.  Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase. , 2003, Carbohydrate research.

[19]  J. Keasling,et al.  Construction of a part of a 3-hydroxypropionate cycle for heterologous polyketide biosynthesis in Escherichia coli. , 2012, Biochemistry.

[20]  J. Sohng,et al.  Production of 3-O-xylosyl quercetin in Escherichia coli , 2013, Applied Microbiology and Biotechnology.

[21]  M. Koffas,et al.  Microbial production of value-added nutraceuticals. , 2016, Current opinion in biotechnology.

[22]  S. Mousa,et al.  Chemoenzymatic Synthesis of Homogeneous Ultralow Molecular Weight Heparins , 2011, Science.

[23]  Ahmad,et al.  Mechanistic Study , 2015 .

[24]  M. Yoshida,et al.  Production of Trehalose from Starch by Maltose Phosphorylase and Trehalose Phosphorylase from a Strain of Plesiomonas , 1997 .

[25]  M. Kurimoto,et al.  Production of Trehalose from Starch by Thermostable Enzymes from Sulfolobus acidocaldarius , 1997 .

[26]  W. R. Farmer,et al.  Improving lycopene production in Escherichia coli by engineering metabolic control , 2000, Nature Biotechnology.

[27]  Qipeng Yuan,et al.  A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate , 2013, Applied and Environmental Microbiology.

[28]  C. Lim,et al.  Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin , 2015, Applied and Environmental Microbiology.

[29]  E. Muñoz-Elías,et al.  Carbon metabolism of intracellular bacteria , 2006, Cellular microbiology.

[30]  Long Liu,et al.  Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor. , 2012, Metabolic engineering.

[31]  J. Zhan,et al.  Efficient glycosylation of puerarin by an organic solvent-tolerant strain of Lysinibacillus fusiformis. , 2014, Enzyme and microbial technology.

[32]  Sang Yup Lee,et al.  Metabolic engineering of Corynebacterium glutamicum for the production of L-ornithine. , 2015, Biotechnology and bioengineering.

[33]  Qipeng Yuan,et al.  Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan. , 2014, ACS synthetic biology.

[34]  H. Zoerb,et al.  Preservation mechanisms of trehalose in food and biosystems. , 2005, Colloids and surfaces. B, Biointerfaces.

[35]  K. Bettenbrock,et al.  Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems , 2012, Journal of bacteriology.

[36]  George H. Kidd,et al.  Trehalose is a sweet target for agbiotech , 1994, Bio/Technology.

[37]  Masaru Kato,et al.  Production of trehalose from starch by novel trehalose-producing enzymes from Sulfolobus solfataricus KM1 , 1997 .

[38]  G. Stephanopoulos,et al.  Optimization of a heterologous pathway for the production of flavonoids from glucose. , 2011, Metabolic engineering.

[39]  Irene M. Brockman,et al.  Improving product yields on D‐glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources , 2014, Biotechnology and bioengineering.

[40]  Jian Chen,et al.  Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli , 2015 .

[41]  N. Costantino,et al.  E. coli genome manipulation by P1 transduction. , 2007, Current protocols in molecular biology.

[42]  Joong-Su Kim,et al.  Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus , 1998, Biotechnology Letters.

[43]  Jingwen Zhou,et al.  Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli , 2015, Scientific Reports.

[44]  G. Stephanopoulos,et al.  Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli. , 2016, Metabolic engineering.

[45]  A. Strøm,et al.  Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression , 1993, Molecular microbiology.

[46]  Chiara Schiraldi,et al.  Trehalose production: exploiting novel approaches. , 2002, Trends in biotechnology.

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

[48]  J. Deutscher,et al.  The mechanisms of carbon catabolite repression in bacteria. , 2008, Current opinion in microbiology.

[49]  Jian Liu,et al.  Design of biologically active heparan sulfate and heparin using an enzyme-based approach. , 2009, Natural product reports.

[50]  Satoshi Ohtake,et al.  Trehalose: current use and future applications. , 2011, Journal of pharmaceutical sciences.

[51]  G. Stephanopoulos.  Challenges in Engineering Microbes for Biofuels Production , 2007, Science.