Construction Of Metabolically Engineered E.coli Producing High Titer Of Pure D-lactate

D-Lactate is an important chiral intermediate as well as a substrate for polylactic acid (PLA)production, where production of both chirally and chemically pure D-lactate from renewableresources using microorganisms is the precondition of the industrial application. Escherichiacoli could ferment glucose to optically pure D-lactate without genetic engineering. However,using wild type E. coli strains, high levels of byproducts are produced in the final broth,resulting in low lactate yield and productivity. Moreover, like the fermentation processes ofother orgnic acid, lactate fermentation is contradictive in the metabolism and regulationprocesses of cell growth and lactate formation. This universal problem is expected to besovled through current industrial microorganism breeding theory and technology to make theprocessing and controlling of lactate fermentation more smoothly and efficiently.In the present study, genetic engineering and metabolic engineering were used toreengineer several competitive routes of D-lactate in a wild type E. coli (CICIM B0013) andto coordinate lactate production rate with cell activity by fine tuning and genetic switching thelactate production route. Recombinant strain producing chirally and chemically pure D-lactatewith high productivity and cell activity was obtained which could be served as a candidate forfull scale production. The main results are as follows:1. Effects of inactivation of ten lactate competitive routes were studied to construct therecombinant E. coli strain with high D-lactate titer and low byproduct level. Afterconstructing the high efficient multiple gene disruption approach in E. coli, competitive routesin D-lactate production such as acetate kinase (ackA), phosphotransacetylase (pta),phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactatedehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), fumaratereductase (frdA), propionate kinase (tdcD) and pyruvate formate lyase4(tdcE) werecumulatively inactivated in B0013. Subsequently, single-gene as well as multiple-geneinactivation of these routes were tested for the effects in two-phase fermentations (aerobicgrowth and oxygen-limited production). Single-gene deletions of ackA, pta, pflB, dld, poxBand frdA can promote lactate yield and productivity; however, single-gene deletions of ppsand adhE had adverse effect on lactate yield and productivity. Cumulative inactivation ofackA-pta, pflB, dld, adhE and frdA were helpful in increasing lactate yield and decreasingbyproducts; whereas, these parameters were not affected by cumulative deletions of pps andpoxB, and acetate level was decreased by cumulative deletion of tdcDE with significantreduction in growth rate. Inactivation of eight genes in B0013to produce B0013-070(ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity to two-foldand reduced byproduct yields of acetate, succinate, formate and ethanol by95,89,100and93%, respectively. While tested in bioreactor, B0013-070produced125g/L D-lactate withincreased oxygen-limited lactate yield of96g/100g glucose and productivity of0.61g/g·h(2.1-fold of that of B0013). A moderate increase in oxygen supply during lactate productionphase further improved lactate productivity in B0013-070to5.5g/L·h, with a chemical purityof98.4%. 2. Fine tuning the transcription of ldhA by shortening its upstream region couldregulate lactate production rate and facilitate high cell activity. ldhA genes with differentlength of upstream regions were cloned in a low copy number vector. The resultingrecombinant plasmids were transferred into B0013-080C (B0013-070, ldhA) to giveB0013-080C/pTH-rrnB-ldhA0~B0013-080C/pTH-rrnB-ldhA11. Fermentations of thesestrains showed that shortening the ldhA upstream sequence from291to106bp successivelyreduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase.Simultaneously, oxygen-limited lactate productivity was increased during the second phase.Putative promoter downstream the-96site of ldhA could function as transcriptional promoteror regulator. No transcriptional promoting region would exist downstream the-61site of ldhA.B0013-080C/pTH-rrnB-ldhA8, with72bp upstream segment of ldhA, could grow with highrate and produce high oxygen-limited lactate productivity of1.09g/g·h.3. A genetic switch was designed to control the transcription of ldhA by regulating thefermentation conditions to dynamically and efficiently regulate cell growth and lactateproduction. Lactate fermentation is contradictive in the metabolism and regulation processesof cell growth and lactate formation. Precise regulating this contradiction using simplefermentation conditions could achieve high efficient cell growth and lactate productionsimultaneously. The λ pRand pLpromoters from pPL451were used to replace the promoter ofchromosomal ldhA (ldhAp) in B0013-070to produce B0013-070B (B0013-070,ldhAp::kan-cIts857-pR-pL). Cultivation conditions for the new strain was: growing cells at33°C and then at42°C for30min followed by switching to the fermentation stage. Inbioreactor fermentation using the scaled-up conditions optimized in shake flask, aerobicbiomass yield in B0013-070B was9%higher than B0013-070. Oxygen-limited lactatevolumetric and specific productivities were improved by51and46%, respectively, with achemical purity of98.6%.