Biosynthesis Of Hydroxypropionate And Its Polyester

Considering the limited deposits of fossil fuels, bio-based platform chemicals, building blocks for numerous chemical intermediates and end products, are recognized as a burning issue in the last decade. Hydroxypropionic acid, consisting of 3-hydroxypropionic acid (3-HP) and 2-hydroxypropionic acid (lactate), was identified as one of the most important platform chemicals. The presence of two functional groups with different properties makes both of them suitable precursor for the synthesis of many optically active substances.3-Hydroxypropionic acid is a chemical reagent well known for its simple structure and its high reactivity.3-HP has utility for specialty synthesis and can be converted to commercially important intermediates by known art in the chemical industry, e.g., acrylic acid by dehydration, malonic acid by oxidation, esters by esterification reactions with alcohols, and reduction to 1,3-propanediol.3-HP for commercial use is now commonly produced by chemical syntheses, its use has remained on a laboratory scale due to its insufficient production, complex separation/purification and higher production costs. At present, the production of 3-HP by genetic engineering and microbial fermentation consists mainly of two parts:I, The genetic engineering progress of producing 3-HP from glucose;Ⅱ, The genetic engineering progress of producing 3-HP from glycerol. However, it's certainly difficult to construct the certain former pathway mentioned above. And the production of 3-HP from glycerol was only 0.17 g/L and unable to meet the requirement for large-scale production.Lactate (2-hydroxypropionic acid) is the most widely occurring hydroxycarboxylic acid, having versatile applications in food, pharmaceutical, textile, and chemical industries. Although several lactic acid bacteria, such as Lactobacillus species, were able to produce lactic acid in a large quantity by fermentation of glucose and other renewable resources, Escherichia coli has many advantages as a host for production of lactic acid, including rapid growth under both aerobic and anaerobic conditions, the ability to produce optically pure lactate, and its simple nutritional requirements. Moreover, the ease of genetic manipulation of E. coli makes possible metabolic engineering strategies for improving lactate accumulation in E. coli. Polylactate (PLA), which is chemically synthesized by ring-opening polymerization of a cyclic diester (lactide) of lactate, has attracted considerable interest as a natural, biodegradable, and biocompatible plastic. However, as the chemo-process of PLA can be carried out via harmful metal catalysts, it often leaves chemical residues that are subject to health and safety concerns. The paradigm shift from the chemo-process to the bio-process for PLA production is thus preferable to overcome this problem.In this article, we constructed a genetic pathway for 3-HP production from glucose in E. coli. A series of recombinants defecting in competitive pathways aiming to produce lactate effectively were obtained. We also established a recombinant E. coli that allows the synthesis of LA-based polyester. The major results of the article are as follows:1. Construction of recombinant E. coli to accumulate 3-HP from glucoseThe initial step of 3-HP cycle in Chloroflexus aurantiacus is the acetyl-CoA carboxylation to malonyl-CoA catalyzed by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-HP. In E. coli, the formation of malonyl-CoA from acetyl-CoA plus CO2 occurs as the first committed step of the fatty acid synthetic pathway catalyzed by the multi-component acetyl-CoA carboxylase (ACCase). The biofunctional malonyl-CoA reductase from C. aurantiacus, encoded by mcr gene, consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain and catalyzes two-step reduction.The mcr gene was PCR amplified from C. aurantiacus strain OK-70-fl (DSM636) and inserted into pET-28a to give plasmid pET-28a-mcr. Recombinant DE3/pET-28a-mcr for production of 3-HP was constructed by transforming E. coli BL21 (DE3) with plasmid pET-28a-mcr. Recombinant DE3/pET-28a-mcr, together with BL21 (DE3)/pET-28a, were inoculated into 50 ml LB medium with 2% glucose and incubated for 60 h. Samples were removed for GC analysis of 3-HP. The retention time of 3-HP by GC is 3.2 min. There is a detectable peak at the same location for the fermentation supernatant of DE3/pET-28a-mcr, corresponding approximately to 0.15 g/L 3-HP.This novel biosynthetic pathways allowed us to achieve the biosynthesis of 3-HP at both the domestic and international level. With the help of metabolism engineering technology, we envision that it will be the solid theoretical basis to make the high yield of 3-HP.2. Construction of a series of recombinants defecting in competitive pathways to produce lactate effectivelyFermentation of sugars through native pathways in E. coli under anaerobic conditions produces a mixture of products consisting primarily of lactate, formate, acetate and ethanol, with smaller amounts of succinate. The relative proportions of these products varied with the relative in vivo enzyme activities such as lactate dehydrogenase (ldhA gene), pyruvate formate lyase (pfl gene) and phosphoenolpyruvate carboxylase (ppc gene). Meanwhile, this product ratio also changed with the growth conditions in order to balance the number of reducing equivalents generated during glycolytic breakdown of the substrate. Acetate and ethanol are typically produced from acetyl-CoA in approximately equimolar amounts, catalyzed by acetate kinase (ackA)/phosphostransacetylase (pta) and alcohol/aldehyde dehydrogenase (adhE) respectively, to provide redox balance.In this study, we constructed recombinant E. coli SD2 and SD4, defecting in pflB and adhE respectively, to improve the production of lactate. Anaerobic fermentation was performed in LB medium supplemented with 100 mM glucose. Deletion of pflB in SD2 obviously increased the lactate production to 174.8 mM, while inactivation of adhE led it to 178.3 mM, approaching the theoretical maximum of 2 mol of lactate per mol of glucose utilized. The mutation of ptsG in SD6 and SD8, derivatives of SD2 and SD4 respectively, altered the fermentative metabolism of E. coli and caused over five fold increase in the formation of succinate at the expense of lactate. Meanwhile, ptsG mutation led to reduced glucose uptake rate but improved biomass during the fermentation.The fermentation products of SD6 and SD8 varied with respect to the different composition of medium. Compared to no more than 16 mM in LB medium after 60 h fermentation, the formation of lactate in SD6 and SD8 with M9 medium largely improved to over 100 mM. Correspondingly, succiante produced by SD6 and SD8 dropped from 45.5 mM,42.5 mM with LB medium to 34.5 mM,31.4 mM with M9 medium, respectively. The existence of potassium ion in M9 was speculated to accounting for the increased lactate conversion. Replacement of potassium with sodium in M9 medium slightly reduced the accumulation of lactate in SD4 and SD8, accompanied by increased succinate production. It's suggested that M9 medium is more conducive to lactate formation than LB.Influences of carbon sources with high degree of reduction, reducing agents, and oxygen availability on the contribution of products were tested. All three approaches expanded the production ratio of lactate to succinate and the dissolved oxygen tension was a key constraint.Through genetic manipulation, high-yield accumulation of lactate was achieved in engineered E. coli SD4 and SD8. Influences of carbon flow and the availability of reducing equivalents on lactate production provided an important technical support for controllable lactate production.3. Construction of a bioprocess for the production of LA-based polyester P(LA-co-3HB)Based on the substrate specificity of PHA synthase, a key enzyme for polymerization of various monomers to polyhydroxyalkanoate (PHAs), which has monomeric constituents share the common chemical structure, hydroxy acid, with 2-hydroxypropionate (the same as LA), we succeeded in creating a microbial biosynthetic system for LA-based polyesters, P(LA-co-3-HB), copolymerized with 3-hydroxybutyrate (3HB), which is a typical constituent of polyhydroxyalkanoates (PHAs). P(LA-co-3HB) is intracellularly synthesized by successive enzymatic reaction steps, as follows:(i) generation of lactyl-coenzyme A (LA-CoA) by propionyl-CoA transferase (PCT) from Clostridium propionicum, (ii) supply of 3-hydroxybutyryl-CoA (3HB-CoA) via the dimerization pathway catalyzed by PhaA (β-ketothiolase) and PhaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, and (iii) copolymerization of the CoA esters by PhaEC, the PHA synthase from Allochromatium vinosum. In our work, a copolymer consisting of 0.22 mol% of LA and 99.78 mol% 3HB was produced in recombinant Escherichia coli DH5a/pBBRl1pcrEC+pBHR69 under aerobic conditions supplied with 1% lactate. Furthermore, LA fraction in the copolymer was increased up to 1.49 mol% by conducting anaerobic culture preferable for LA production.Construction of this engineered system is the first of its kind in this country to make up the domestic technology gap in PLA production. It plays an important role in helping to understand the bio-process synthesis of PLA, thus has important theoretical significance and application value.