Study On In Vitro Synthetic Biology Based On Glycerol And Lactic Acid

With the dwindling of oil resources and the increasingly growing environmental concern,the biofuel industry is developing rapidly.The large surplus of glycerol derived from the expanding biofuel industry may cause economic and environmental concerns with regard to glycerol disposal.Conversion of glycerol into high-value products is of significant importance for the sustainable development of the biofuel industry.Compared with chemical catalytic processes,biocatalysis has many advantages,such as high stereoselectivity,mild reaction conditions and low pollution.Because of the complexity and diversity of intracellular metabolic pathways,it often leads to the production of unwanted by-products,resulting in decline in conversion efficiency and yield of target products.One of the possible solutions for the above-mentioned problem is to use a cell-free bio-system,which leaves out the cells and exclusively employs purified enzymes.Compared with the traditional fermentation processes,the in vitro synthetic biosystems are superior in many aspects,such as fast reaction rate,high yield of target products,strong tolerance to toxic substances and flexible design.In this study,pyruvic acid,a central intermediate needed for the production of versatile biomolecules,was produced from glycerol without the addition of any cofactors by the cell-free bio-system composed of alditol oxidase from Streptomyces coelicolor A3(ALDO),dihydroxy acid dehydratase from Sulfolobus solfataricus(DHAD),and catalase from Aspergillus niger.Pyruvic acid was produced at a yield of 0.93 mol/mol of the theoretical yield.Since Pyruvic acid was an important intermediate metabolite,it was readily to be converted into valuable chemicals Herein,the production of acetoin from glycerol was carried out through the coupling of ?-acetolactate synthase(ALS)and ?-acetolactate decarboxylase(ALDC)from Bacillus licheniformis 10-1-A.Acetoin was produced at 85.5%of the theoretical yield from glycerol.The ee value of(3R)-acetoin was estimated to be 95.4%.By using rational assembly of thermostable enzymes from various species,the artificially designed in vitro biosystem was further developed to bimanufacture other valuable synthetic building blocks that are of different reduction degrees.By coupling of tyrosine phenol-lyase from Symbiobacterium thermophilum(StTYRPL),condensation of glycerol,phenol,and ammonium into L-tyrosine was achieved.The in vitro biocatalytic process was performed using only four enzymes without the assistance of NAD+/NADH-related redox reactions.At 24 h,the yield of L-tyrosine was 77.1%.The ee value of L-tyrosine was measured to be more than 99.9%.To produce chemicals of high reduction degrees from glycerol,coupling of the NADH recycling system is technically feasible and easy to implement.Herein,biotransformation of glycerol into optical L-lactate and D-lactate was carried out to verify the feasibility of such a system.Formate dehydrogenation was selected for NADH regeneration since it did not introduce unwanted byproducts,thereby simplifying the purification of the target products.In this study,the formate dehydrogenase in Ogataea parapolymorpha DL-1(OpFDH)was selected since it possesses promising thermostability,a wide pH optimum,and high organic tolerance D-Lactate dehydrogenase from Thermodesulfatator indicus(TiDLDH)and L-lactate dehydrogenase from Thermus thermophilus HB8(TtLLDH)was selected for production of D-lactate and L-lactate,respectively.L-Lactate at an ee value of 100%was produced from glycerol at 24 h at a yield of 91.6%.D-Lactate at an ee value of 100%was produced glycerol at a yield of 97.3%.To explore the advantages of glycerol further and eliminate exogenous NADH regeneration,we designed a completely artificial pathway for conversion of glycerol into L-serine that was composed of only four enzymes,thereby also eliminating the reactions catalyzed by ATP or ADP.The glycerate generated from glycerol oxidation was converted into 3-hydroxypyruvate under the catalysis of NAD+-dependent glyoxylate/hydroxypyruvate reductase.The substrate promiscuity of alanine dehydrogenase was exploited to catalyze the reductive amination of 3-hydroxypyruvate into L-serine.The glyoxylate/hydroxypyruvate reductase from the hyperthermophilic archaea Pyrococcus furiosus(PfuGRHPR)and the L-alanine dehydrogenase from another hyperthermophilic archaea Archaeoglobus fulgidus(AfALADH)were selected.L-Serine at an ee value of more than 99.9%was produced from glycerol at a yield of 71.3%at 18 h.The biotransformation of glycerol and ammonium into L-serine was achieved using four enzymes with self-sufficient NADH recycling.These properties of the in vitro biosystem make it a versatile and useful platform for production of value-added chemicals from glycerolThe increasing world population is leading to an increasing demand for food supply,resulting in the generation of large amounts of polluting organic waste from the food industry.Corn wet milling is a significant industrial activity,but it produces a large amount of corn steep water that is characterized by high waste load.One of the economically viable processes is separating important chemicals from corn steep water.Lactate is one of the most abundant chemicals in the corn steep water with content of about 20-50 g/L.Lactate can be separated from corn steep water with a rather low cost,which supports the value-added utilization of corn steep water.However,numerous lactate-producing Lactobacillus strains participate in lactate production in the corn steeping process,resulting in the production of both D-lactate and L-lactate during the corn steeping process.The racemic characteristic of lactate from corn steep water restricts the development of this promising process.In this study,based on D-lactate oxidase(D-LOX)from Gluconobacter oxydans,L-lactate oxidase(L-LOX)from Pediococcus sp.,pyruvate decarboxylase(PDC)from Zymomonas mobilis and catalase from bovine liver,we synthesized an in vitro bio-system,including different enzymatic cascades,for the production of valuable platform chemicals from lactate separated from the corn steep water.Using these enzymatic cascades constructed by the enzymes mentioned above,C2(acetaldehyde),C3(D-lactate,L-lactate and pyruvate),and C4(acetoin)platform chemicals can be produced with high yields.In the in vitro biosystem for production of L-lactate and pyruvate,pyruvate was produced with a yield of 96.7%.L-Lactate with an ee value>99%was produced at a residual rate of 98.2%.In the biosystem for production of D-lactate and pyruvate,pyruvate was produced with a yield of 99.2%.D-Lactate with an ee value>99%was produced at a residual rate of 99.7%%.In the biosystem for production of pyruvate,pyruvate was produced with a yield of 97.9%.In the in vitro biosystem for production of L-lactate and acetaldehyde,acetaldehyde was produced with a maximum yield of 73.8%.L-Lactate with an ee value>99%was produced at a residual rate of 97.7%.In the biosystem for production of D-lactate and acetaldehyde,acetaldehyde was produced with a maximum yield of 74.8%.D-Lactate with an ee value>99%was produced at a residual rate of 97.3%.In the biosystem for production of acetaldehyde,acetaldehyde was produced with a maximum yield of 66.9%.In the in vitro biosystem for production of L-lactate and acetoin,acetoin was produced with a yield of 88.4%.L-Lactate with an ee value>99%was produced at a residual rate of 99.7%.In the biosystem for production of D-lactate and acetoin,acetoin was produced with a yield of 86.7%.D-Lactate with an ee value>99%was produced at a residual rate of 99.9%%.In the biosystem for production of acetoin,acetoin was produced with a yield of 92.7%.Since pyruvate has the potential to be converted into various molecules,more value-added platform chemicals might also be produced from racemic lactate through further development of other biotransformation systems.The in vitro bio-system not only provides a novel technology platform for the production of optical lactate and lactate derivatives,but also might support the sustainable development of corn starch industry.5-Aminovalerate is the precursor for the synthesis of nylon-5.5-Aminovalerate x could also serve as a potential C5 platform chemical to produce 5-hydroxyvalerate,glutarate,and 1,5-pentanediol et al.5-Amniovalerate could be produced through biotechnological routes,which guarantees the green route to nylon-5 production.In this study,lysine 2-monooxygenase(DavB)and ?-aminovaleramidase(DavA)from Pseudomonas putida KT2440 were co-expressed in Escherichia coli BL21(DE3)to produce 5-aminovalerate from L-lysine.Since bacterial transport proteins mediate the exchanges between intracellular and extracellular environments,modification of transport route could be applied to speed up the metabolic reactions and promote the production of aimed compounds.In this regard,PP2911(4-aminobutyrate transporter in P.putida KT2440)and LysP(the lysine specific permease in E.coli K12)were overexpressed to promote 5-aminovalerate production using whole cells of the recombinant E.coli pDABLP.The bioconversion of L-lysine into 5-aminovalerate was conducted under optimized conditions.29.6 g/L 5-aminovalerate was produced from 38.6 g/L L-lysine in 48 h at a yield of 0.96 mol/mol.Then the fed-batch process was performed to evaluate the production capacity of the recombinant E.coli pDABLP.At 156 h,63.2 g/L 5-aminovalerate was produced from 102.3 g/L L-lysine at a yield of 0.77 mol/mol.The constructed E.coli strain overexpressing transport proteins exhibited good 5-aminovalerate production performance and might serve as a promising biocatalyst for 5-aminovalerate production from L-lysine.This strategy not only introduced an efficient process for production of nylon monomers,but also might be used in the production of other chemicals.