Conversion Of Glycerol To Lactic Acid With Metabolically Engineered Escherichia Coli

Currently, petrochemicals are used as raw materials in the manufacturing of a variety ofproducts such as polymers, textiles, paints and solvents etc. However, rapid depletion of thepetroleum resource and increase in emission of greenhouse gas encouraged a replacement ofpetroleum with renewable resources. Polylactic acid, as typical biodegradable material, hasshowed a potential to replace petrochemical-based plastics. Manufacturing polylactic acidneeds large amounts of monomer, D-lactic acid and/or L-lactic acid. Therefore,biotechnological production of D-lactic acid and/or L-lactic acid with high optical purity andchemical purity has been increasingly focused in recent years. Production of lactic acid fromcheap available biomass was investigated extensively. Glycerol is the most readily availablerenewable feedstock. With the increasing international biodiesel production, a surplus ofcrude glycerol has been generated resulting in a significant price reduction. In addition to lowcost and abundance, the higher degree of reduction makes glycerol be an excellent potentialcarbon source to produce chemicals with high yield.In this study, metabolic engineering and process optimization were employed toinvestigate bioconversion of glycerol into D-lactic acid and L-lactic acid by Escherichia coli.The main research findings are as follows:1. Efficient production of D-lactic acid from glycerol was achieved through geneticmodification of the metabolic pathway and optimization of the fermentation process.The fermentation process for D-lactate production from glycerol was optimized byincreasing the oxygen supply strength during lactate formation phase. In7-L bioreactor andusing B0013-070strain,98.5g/L of the final concentration and3.45g/L of productivity ofD-lactate were generated, respectively. Furthermore, increasing copy of ldhA gene encodingD-lactate dehydrogenase led to higher production of D-lactic acid, and the final D-lactic acidconcentration and productivity were103.1g/L and3.65g/L h, respectively. The yield reached78.0%.Moreover, temperature was optimized during cell growth phase and lactate productionphase, respectively. The resulting showed that34°C was optimal for cell growth and42°Cwas optimal for production of lactic acid. Using the temperature-shifting process, engineeredstrain B0013-070produced D-lactic acid with a3.66g/L h of productivity and82.6%of yield.Using another strain E. coli B0013-070B in which the promoter of ldhA was replaced bytemperature-inducible promoter, production performance of D-lactic acid further increased bythis fermentation process. And4.23g/L h of productivity and88.9%of yield were obtained.2. Metabolic engineering of E. coli for efficient production of L-lactate from glycerolAn exogenous L-lactate dehydrogenase gene (BcoaLDH) was cloned from thermophilicBacillus coagulans CICIM B1821and expressed in E. coli to achieve L-lactic acid producerfrom glycerol. To further increase L-lactic acid production, the BcoaLDH fusing promoter ofldhA was inserted into E. coli chromosome and replacing of the lldD gene that encodes FMN-dependent L-lactate dehydrogenase catalyzing L-lactic acid to pyruvate. Furthermore,the D-lactic acid synthesis pathway was blocked by deleting the ldhA gene to realize extremelyhigh optical purity L-lactic acid synthesis. The resulting strain B0013-090B was used toevaluate the production of L-lactic acid. Using temperature-shifting process,132.4g/L ofL-lactic acid was produced from glycerol, and4.90g/L h of productivity and93.7%of yieldwere obtained, respectively. The optical purity of L-lactic acid reached to99.9%.3. Fine switch of cell growth and latctic acid production using thiaminehydrochloride through deletion of thiE gene producing thiamine auxotrophy phenotype.And the concise fermentation process of engineered strain was developed for D-lactateproduction.The thiE gene encodes a critical protein in the synthesis route of thiamine, a cofactor forPDH complex. Therefore, deletion of thiE gene produced thiamine auxotrophy phenotype.We deleted the thiE gene in the D-lactate producer Escherichia coli CICIM B0013-070(ackA, pta, pps, pflB, dld, poxB, adhE, frdA) to generate CICIM B0013-080A.The cell mass of B0013-080A was fine cotrolled by the concentration of thiaminehydrochloride (VB1) added into medium. The equation of the relationship between theamount of VB1and the biomass was obtained as follows: y=(4.0×1010)x+1.5902(R2=0.9987),where y is the obtained biomass and x is the amount of thiamine hydrochloride (VB1) added.When VB1was added into the fermentation medium with a final concentration of2.47×10-7μg/mL, engineered strain performed better for cell growth and D-lactic acid synthesis. Thefinal concentration of D-lactic acid reached119.3g/L. The productivity and yield of D-lactatefrom glycerol increased to4.06g/L h and87.1%, respectively. Furthermore, when theD-lactate dehydrogenase temperature-inducible transcription system was introduced to theauxotrophic strain, efficiency of D-lactate production improved significantly. Usingtemperature-shifting process, the final concentration of D-lactic acid reached129.3g/L. Theproductivity and yield of D-lactate from glycerol increased to4.79g/L h and92.1%,respectively.