1, 3-propanediol Production From Different Material And Construction Of Recombinant Strains

1,3-propanediol (1,3-PDO) is one of the most interested material for chemical industries due to its wide use in different fields such as food, polymers and medical applications. For example, one of the most successful application for 1,3-PDO has been in the synthesis of polytrimethylene terephthalate (PPT) that is wildly used in the manufacture of carpet an textile fibers. Annually over one million ton of 1,3-PDO is produced by chemical method, which requires high cost and is non-friendship to the environment. Most interests have now been focused on the production of 1,3-PDO from the renewable resources through economical and "green" processes such as microbial fermentation. However, in nature a few microorganisms can produce 1,3-PDO, but they all use an expensive resource pure glycerol as substrate, this production mode is not still competitive with chemical synthetic process. In order to promote efficiency or reduce the material cost of 1,3-PDO production, in this study, we studied on the 1,3-PDO producer Klebsiella pneumoniae, model microorganism Saccharomyces cerevisiaeW303-1A and glycerol producer strain Candida glycerinogenes, respectively, using metabolic engineering, molecular biotechnology and fermentation biotechnology. The main contents of this dissertation are as following:1,In order to reduce the accumulation of intermediate 3-hydroxypropionaldehyde (3-HPA), which roles as an inhibitor during the process from glycerol to 1,3-PDO in K. pneumoniae, we constructed the novel expression system for K. pneumoniae based on plasmid pET28a carrying constitutive expression promoter Pkan. The transcription activity of promoter Pkan in K. pneumoniae was proved by importing the chloramphenicol acetyltransferase gene(cat). Further, this expression system was successfully applied to the over-expression of gene dhaT encoding PDOR for reducing of 3-HPA accumulation and increasing of 1,3-PDO production in the recombinant strains K. pneumoniae/pETPkan-dhaT. The enzyme PDOR was over-expressed, and the recombinant K. pneumoniae/pETPkan-dhaT showed a nearly 3.0-fold decrease in peak level of the intermediary metabolite 3-HPA and an increase of 16.5%in yield of 1,3-PDO with respect to the wild-type strain K. pneumoniae.2,In order to further improve the 1,3-PDO yield of K. pneumoniae/pETPkan-dhaT, the effects of nutritional (initial glycerol concentration, nitrogen source,) and environmental condition (pH, temperature, inoculum) on conversion rate of glycerol to 1,3-PDO and cell growth were investigated in shaking flask. The optimized conditions were determined by using orthogonal experiments as follows:60 g/L glycerol as carbon substrate,7 g/L yeast extract and 0.3 g/L (NH4)2SO4 as nitrogen source,8 g/L citric acid. The optimum initial pH, culture temperature, initial liquid volume and inoculums for 1,3-PDO production was 7.0,30℃,50 mL/250mL flask,6%, respectively. Under optimized fermentation conditions, the 1,3-PDO yield reach up to 37.7 g/L and the conversion rate of glycerol to 1,3-PDO can reach 62.8%. The effects of different stir rotation speeds on the fermentation in 7 L fermentor were studied. In the 300 r/min condition, the 1,3-PDO concentration and conversion rate were 36.4. g/L,60%, respectively. Furthermore, the fed-batch fermentation results showed that the production of 1,3-PDO was yielded 62.2 g/L after 45 h in 7 L fermentor.3,The different glycerol types derived from C. glycerinogenes were used as substrate for 1,3-PDO production by K. pneumoniae/pETPkan-dhaT. The glycerol broth from industrial strain C. glycerinogenes was first used as the sole carbon source, the culture broth from C. glycerinogenes was shown to contain toxic metabolites that strongly impair the growth of K. pneumoniae/pETPkan-dhaT and glycerol consumption rate. The 1,3-PDO yield was only 8.9 g/L. However the C. glycerinogenes broth acetate and ethanol formation can be reduced by limiting glucose supply and adding NaCl to keep osmotic stress condition, this broth was used as substrate to produce the 1,3-PDO by recombinant strain, the 1,3-PDO yield can be increase to 24.7 g/L. Finally, the raw glycerol was prepared by extraction and purification, the effect of raw glycerol on 1,3-PDO production was also investigated. The 1,3-PDO yield and conversion rate were 33.5 g/L and 55.8%, respectively. These results showed that raw glycerol could be directly utilized in shake flask fermentation by K. pneumoniae with similar results obtained from pure glycerol. Further, the fed-batch fermentation using raw glycerol as substrate, the 1,3-PDO could be produced 57.8 g/L. The above results demonstrated that the raw glycerol as a low-cost materail replaced pure glycerol was suitable for 1,3-PDO production.4,8.0 kb dha operon was amplified from K. pneumoniae and ligated into pMD18-T vector, then the recombinant plasmid was transformed into Escherichia coli JM109, the resulting transformant E. coli JM109 characterized for activities of GDHt and PDOR and the ability to convert glycerol to 1,3-PDO which also confirmed that the dha operon was required for the synthesis of 1,3-PDO from glycerol. The dha operon was subsequently ligated into vector pYX212-zeocin and directly expressed in the S. cerevisiae, The yield of 1,3-PDO in this recombinant S. cerevisiae could not be detected due to the low level of enzymatic activity. To enhance the enzymatic activities of GDHt and PDOR in S. cerevisiae, we planned to over-express genes dhaB and dhaT under the control of regulatory component from yeast in S. cerevisiae W303-1A. Both genes dhaT and dhaB were cloned into the vector pGAPZB between the promoter GAP (glyceraldehyde 3-phosphate dehydrogenase) and AOXITT terminator to form plasmid pZ-BT. The recombinant strain W303-BT can directly produce the 1,3-PDO from glucose. The enzymatic activities of GDHt, PDOR and 1,3-PDO yield in our recombinant S. cerevisiae were 15.1 U/mg,12.3 U/mg and 1.2 g/L, respectively.5,We cloned the cassettes dha1 and dhd2 required for the 1,3-production from glycerol, and placed them under the control of promoter GAP to insert into binary vector pCAMBIA3300-zeocin to create plasmid pGAP4, then integrated them in the chromosome of C. glycerinogenes via Agrobacterium tumefaciens-mediated transformation (ATMT). The integration of both cassettes dha1and dha2 in the chromosome of recombinant yeast strain WL2002-GAP was further confirmed by PCR and Southern blot. The enzymatic activities of GDHt, PDOR and 1,3-PDO yield from glucose in WL2002-GAP were 1.9 U/mg,1.4 U/mg and 2.9 g/L, respectively.6,The use of homologous salt stress-inducible promoter Pcggpdl is expected to be optimal for driving the expression of dha genes in C. glycerinogenes. The promoter GAP in the plasmid pGAP4 was replaced by promoter Pcggpdl to create plasmid pGPD4. Then the expression of cassettes dhal and dha2 were driven by Pcggpdl, and integrated into the chromosome of C. glycerinogenes by ATMT to construct the engineered WL2002-GPD. The enzymatic activities of GDHt and PDOR in WL2002-GPD were 5.1 and 3.7 U/mg, which showed an increase of nearly 2.7-fold and 2.6-fold higher than those in WL2002-GAP, respectively. The 1,3-PDO yield from glucose was 6.7 g/L, which was 2.3-fold higher than the value in WL2002-GAP.7,The engineered WL2002-GPD were grown in the fermentation medium supplemented with 0.5,1 M NaCl. As we expected, the engineered WL2002-GPD can enhance the specific activities and 1,3-PDO concentration in the salt stress-inducible condition with respect to obtained values of WL2002-GPD in the no salt stress-inducible condition. When engineered WL2002-GPD was exposed to salt stress with 0.5 M NaCl, the 1,3-PDO concentration was 7.4 g/L and the specific activities of GDHt and PDOR was reach up to 5.6 U/mg and 4.1 U/mg, respectively, which was 10%higher than those of WL2002-GPD under the no salt stress-inducible condition. When the NaCl concentration of fermentation medium was increased to 1M, the specific activities and 1,3-PDO yield showed an increase of 12.5%and 9.4%, respectively compared to the values of WL2002-GPD in the fermentation condition with 0.5 M NaCl treatment.