Studies On 5-Aminoleyulinate Production Via Recombinants Optimization And Process Design

5-Aminolevulinic acid(ALA) is generally known as an essential intermediate in the biosynthesis of tetrapyrroles such as heme,porphyrin,chlorophyll,and vitamin B₁₂.Recently,ALA has received wide attention as a selective and biodegradable herbicide or insecticide in agriculture.Also ALA as photosensitizer for photodynamic therapy(PDT) and diagnosis is of potential use for cancers,oral errucous hyperplasia and so on.ALA can be produced by fermentation with mutants of photosynthetic bacteria and recombinant organisms.Among them,metabolic engineering technology offers a new choice to increase the expression of ALA and to enhance the productivity of ALA,and will be a promising biotechnology for ALA mass production in the near future.But ALA production with engineered strains is still costly due to the relatively low ALA accumulation and yield.Also the fermentation technique should be improved further for ALA mass production and potential application.The aim of this work is to construct highly efficient recombinant Escherichia coli and develop new and effective approaches for fermentation process to enhance the ALA productivity,decrease production cost and become a promising biotechnology for ALA industrial production and application in agricultural and medical field via metabolic engineering and process design.Firstly,the ALA synthase(ALAS) gene(hemA) was cloned from the genome of Rhodobacter sphaeroides and recombinant plasmid pET28a(+)-Rs.hemA was constructed.Then,succinate dehydratase gene(sdhAB) in chromosome of E.coli MG1655 was knockout to obtain engineered strain MG1655(sdhAB^-) by using the Red recombinantion system with homologous PCR products.And E.coli MG1655 and MG1655(sdhAB^-) were lysogenized and recombinants E.coli MG1655(sdhAB^-) (DE3)/pEY28a(+)-Rs.hemA and MG1655(DE3)/pET28a(+)-Rs.hemA were prepared for ALA production..Since initial glucose,glycine and succinate were observed to be the key factors in the medium,the main interactions among them for ALA production were studied by response surface analysis.Based on the results from shake flask experiments,E.coli MG1655(sdhAB^-)(DE3)/pET28a(+)-Rs.hemA was a better ALA producer.ALA production was achieved to 2.1 g/l,and molar yield(ALA/succinate) was improved to 40.1%under the appropriate conditions in 15-1 fermenter.ALA dehydratase(ALAD) gene(hemB) of E.coli BW25113 was successfully replaced by Thermus thermophilus HB8's via pKD46 mediated Red recombination technology.A metabolic engineering strain with low ALAD activity was obtained.But considering ALAS activity was still low in E.coli BW25113 and MG1655,it's a preference to optimize ALAS.Then ALAS gene containing several codons rarely used by E.coli was optimized in two strains of E.coli:BL21(DE3) and Rosetta(DE3) which is a rare codon optimizer strain.The effects of initial concentration of isopropyl-β-D-thiogalactopyranoside(IPTG),induction time,and temperature on enzyme activity were studied and compared for two strains.The results indicated that the ALAS expressed by Rosetta(DE3)/pET-28a(+)-Rs.hemA was higher than that by BL21(DE3)/pET-28a(+)-Rs.hemA.ALAS activity was generally higher with Rosetta(DE3) than with BL21(DE3),so was ALA biosynthesis.Based on the optimal culture system using Rosetta(DE3),the yield of ALA achieved 3.8 g/l(29 mM) under the appropriate conditions in 5-1 fermenter.ALA production with recombinant E.coli Rosetta(DE3)/pET28a(+)-Rs.hemA was studied in detail.In batch fermentation,the addition of glucose and glycine was effective to improve ALA production.Then the fed-batch fermentation was conducted with continuous feeding of precursors.When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l,respectively,in the feeding,the ALA yield reached 4.1 g/l.But the molar yield(ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation.And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production.A two-stage pH value controlling strategy was suggested,in which,the pH value in the first 6 h was regulated at pH 5.9,after then at pH 6.2,and the ALA yield was as high as 6.6 g/l via fed-batch fermentation.To enhance the expression of ALAS gene from Agrobacterium radiobacter zju-0121,E.coli Rosetta(DE3) was picked out as the host to construct an efficient recombinant strain according to the above results.Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under the appropriate conditions.The results indicated that the activity of ALAS expressed in Rosetta(DE3)/pET-28a(+)-Ar.hemA was about 20%higher than that in E.coli BL21(DE3).Based on an optimal fed-batch culture system described in our previous work,up to 6.5 g/l(50 mM) ALA was produced in a 15-1 fermenter.Then ALAS from E.coli Rosetta(DE3)/pET-28a(+)-Ar.hemA was purified by affinity purification on Ni-NTA agarose and gel filtration chromatography on Sephadex G-25 Medium resin.The recombinant ALAS was characterized,and D-glucose and D-xylose were selected as effective inhibitor for ALAD from E.coli Rosetta(DE3).Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture with Rosetta(DE3)/pET28a-Ar.hemA,the yield of ALA achieved was 7.3 g/l(56 mM) under the appropriate conditions in the fermenter.