Conversion of d-glucose to l-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions

Abstract

Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates. Here, a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of d-glucose to l-lactate via pyruvate. This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation. After optimization of enzyme loading amounts, buffer concentration and cofactor concentration, d-glucose was converted to l-lactate with a product yield of ∼90%. Our study has provided an emerging platform with potentials in producing pyruvate-derived chemicals, and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid

“…Using several HPLC setups and conditions did not result in a clear separation of all intermediates (Figure S4). This has been shown previously to be a challenging task for similarly polar organic acids (Xie et al, 2018). To address this challenge, we performed reactions with 13 C labeled substrate in combination with nuclear magnetic resonance (NMR) spectroscopy to trace every carbon during the reaction in the system.…”

“…Stopping one-pot reactions with 12.5% TCA and analysis on an Agilent Hi-Plex H + as performed in residual activity and sequential pathway tests, led to unsatisfactory results for prolonged one-pot reactions. Instead a different method to precipitate proteins was applied that has been successfully used before (Xie et al, 2018). To stop reactions, 35 μl of 1.9 M HCIO 4 was added to 65 μl of each sample.…”

Engineering of a thermophilic dihydroxy-acid dehydratase to enhance its dehydration ability on glycerate to pyruvate and its application in in vitro synthetic enzymatic biosystems

“…The plasmids pET28a(+)-SsDHAD containing the SsDHAD encoding genedhad from S. solfataricus (Genebank accession number: AKA78631.1), pET28a(+)-TaALDH containing the TaALDH encoding genealdh from Thermoplasma acidophilum (Genebank accession number: CAC11938.1), pET28a(+)-SsGDH containing the SsGDH encoding genegdh from S. solfataricus (Genebank accession number: SSO3003), pET28a(+)-SaKDGA containing SaKDGA encoding gene kdga(Genebank accession number: Saci0225), pET20b-TmLDH containing the TmLDH encoding gene l-ldh from Thermotoga maritima (Genebank accession number: TM1867), and pET20b-TsFDH containing the TsFDH encoding gene fdh from Thiobacillus sp. KNK65MA (Genebank accession number: fdh65MA) were obtained from the previous works (Song et al, 2019;Xie et al, 2018). The plasmid pETduet-SsALDO containing the ScALDO encoding gene aldo from Streptomyces coelicolor A3 (Genebank accession number: NP 630252) was constructed as described elsewhere (Li et al, 2018).…”

“…For example, a thermostable DHAD from hyperthermoacidophilic archaeon Sulfolobus solfataricus (SsDHAD) exhibits high activity on converting 2,3-dihydroxyisovalerate to 2-oxoisovalerate (47 U/mg) (Carsten et al, 2015;Kim & Lee, 2006;Sperl, Carsten, Guterl, Lommes, & Sieber, 2016) and very low activity on converting glycerate to pyruvate (8 mU/mg). The extreme low activity of DHAD on glycerate hinders the production of bio-based products and biofuels by many in vitro synthetic enzymatic biosystems based on non-phosphorylative Entner Doudoroff (np-ED) pathway (Ahmed et al, 2005;Xie et al, 2018). Even though the dehydratation activity of SsDHAD was improved by 1.5-times through non-rational design protein engineering (Begander, Huber, Döring, Sperl, & Sieber, 2020), still cannot meet the requirement of further application.…”

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity

“…Pyruvate was then further converted to produce ethanol and isobutanol. The core of this cascade has recently been adopted and modified to also produce other products like D-lactate and L-alanine [16,17]. Besides starting from D-glucose, the dehydratation of D-glycerate is also a critical step for cascades that convert glycerol into amino acids, for example [18].…”