Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway

Abstract

BackgroundThe integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.ResultsA chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.ConclusionsIn this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

A high-energy-density sugar biobattery based on a synthetic enzymatic pathway

“…13), the inherently low, but Supplementary Table S3. promiscuous, activities of one enzyme that catalyses several substrates results in very low power densities. The use of more than ten enzymes for implementing complex reactions for the production of biocommodities, fine chemicals and pharmaceuticals seems to be not economically prohibitive 25,26,[30][31][32][33] .…”

A synthetic biochemistry molecular purge valve module that maintains redox balance

“…Traditionally, in vitro pathway construction has been relegated to use as a research tool or in applications that require only 1-3 enzymes for the production of chiral compounds and other highvalue chemicals 1,8,9 . Improvements in protein expression and access to stable enzymes have made more complex systems possible, however 1,[9][10][11][12] .…”

In vitro conversion of glycerol to lactate with thermophilic enzymes

“…The expression vector encoding the glycerol kinase of Thermococcus kodakarensis (GK Tk , gi| 3986088) was donated by Dr. Y. Koga, Osaka University [21]. Sources of expression vectors for triose phosphate isomerase (TIM Tt , gi| 3169211), enolase (ENO Tt , gi| 55979971), pyruvate kinase (PK Tt , gi| 55979972), lactate dehydrogenase (LDH Tt , gi| 55981082) of Thermus thermophilus, nonphosphorylating GAP dehydrogenase of T. kodakarensis (GAPN Tk , gi|57640640), and cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii (iPGM Ph , gi| 14589995) were described previously [5]. The expression vector for G3P dehydrogenase of T. thermophilus (G3PDH Tt , gi|55981709) was obtained from the Riken T. thermophilus HB8 expression plasmid set [22].…”

“…A possible solution to this problem is to avoid the use of living microorganisms and to construct an in vitro artificial metabolic pathway in which only a limited number of enzymes are involved. Until now, a variety of in vitro synthetic pathways have been designed and constructed for the production of alcohols [3,4], organic acids [5,6], carbohydrates [7], hydrogen [8,9], bioplastic [10], and even electricity [11]. Particularly, employment of enzymes derived from thermophiles and hyperthermophiles enables the simple preparation of catalytic modules with excellent selectivity and thermal stability [5,12].…”

“…Until now, a variety of in vitro synthetic pathways have been designed and constructed for the production of alcohols [3,4], organic acids [5,6], carbohydrates [7], hydrogen [8,9], bioplastic [10], and even electricity [11]. Particularly, employment of enzymes derived from thermophiles and hyperthermophiles enables the simple preparation of catalytic modules with excellent selectivity and thermal stability [5,12]. Furthermore, although the detailed mechanisms remain to be clarified, many thermophilic enzymes have also been reported to display higher tolerance towards denaturants such as detergents and organic solvents than their mesophilic counterparts [13,14], and activities of some thermophilic enzymes are even improved with organic solvents [15].…”