Construction And Optimization Of Engineering Bacteria For The Direct Production Of2-keto-L-gulonic Acid

2-Keto-L-gulonic acid is an important precursor of vitamin C. The conventional two-stepfermentation route is the most successful method for L-AA production, and has been used onan industrial scale for several decades. However, unlike most of the common biotechnologicalprocesses, the conventional two-step fermentation of L-AA involves three microorganismsand requires an additional second sterilization process. This significantly increases the cost ofboth raw materials and energy requirement. Furthermore, the mix-culture system composed ofBacillus megaterium and Ketogulonicigenium vulgar makes both strain improvement andprocess optimization difficult. Therefore, a one-step fermentation process is considered to bemore cost-effective and revolutionary in the L-AA industry worldwide. In this paper, thecrtical genes were introduced into Escherichia coli and Gluconobacter oxydans respectivelyto construct engineered strains for one-step fermentation of2-KLG, and the production of2-KLG was effectively improved by the use of connecting peptide and the trimeric proteinCutA?and further by the enhancement of the cofactor PQQ. The main results are listed asfollowing:(1) Cloning and identification of2-KLG biosynthetic genesK. vulgare WSH-001is an industrial strain used for vitamin C production. Based ongenome sequencing and pathway analysis of the bacterium, some of dehydrogenases relatedto2-KLG synthesis were predicted, including KVU_PA0245, KVU₂142, KVU₂159,KVU₁366, KVU₀203, KVU₀095and KVU_pmdB₀115. In order to validate theenzymatic properties, these seven putative dehyrogenases were overexpressed in E. coli BL21(DE3) and purified for characterization. It was found that the seven dehydrogenases are PQQdependent enzymes, KVU₁366, KVU₀203, KVU₂142, KVU₂159and KVU_PA0245possess both SDH and SNDH activities, in which KVU₂142has the highest specific activity,9.68U/mg. While KVU_PB0115and KVU₀095only possess SNDH activity, and thespecific activity of the latter is nearly twice that of the former. Furthermore, enzyme kineticparameters were determined, KVU₂142and KVU₀095has the highest substrate bindingability in five SDH/SNDH and two SNDH, respectively. In addition, determination of thecatalytic ability in vitro showed that there was2-KLG generated when L-sorbose wascatalyzed with SDH/SNDH alone or in combination with SNDH, in which the highest2-KLGconcentration was19.7g·L–1with the combination of KVU₂142and KVU₀095.(2) Construction of E. coli engineered strain for the direct production of2-KLGThe tolerance of E. coli to2-KLG was determined, it showed that even100g·L–1of2-KLG did not significantly affect the cell growth of E. coli. In addition, no obviousdegradation of2-KLG could be detected when E. coli was grown with2-KLG for120h. Theengineered strain E. coli/pET-k2142-k0095-pRSF-sldh-pqq was constructed with twoplasmids pETDuet-1and pRSFDuet-1. It was proved that four enzymes were expressedthrough SDS-PAGE detection. In addition, the crude enzyme was used in catalytic reaction,and2-KLG was generated after sorbitol was catalyzed. After72h fermentation of theengineered strain,4.8g·L–1of sorbose,0.5g·L–1of2-KLG and a great deal of by-products was generated. Sorbitol may be transferred into the glycolysis and the pentose phosphatepathway through sorbitol phosphotransferase system.(3) The whole genome sequencing and the construction of gene manipulation system ofG. oxydans WSH-003The whole genome sequence of G. oxydans WSH-003was released by Illuminasequencing technology. The genomic feature was provided in this dissertation. The completeG. oxydans WSH-003genome is3,364,884bp and contains3705protein-encoding genes, theoverall G+C content of the chromosome is50.42%. G. oxydans has a very strong incompleteoxidation ability of carbohydrates, alcohols and polyols. In G. oxydans WSH-003, there areabout330genes involved in the carbohydrate metabolism, and the presence of more than300membrane transport proteins make G. oxydans WSH-003possess a very high conversionefficiency. In addition, the tolerance of G. oxydans WSH-003to2-KLG was determined, itshowed that even100g·L–1of2-KLG did not significantly affect the cell growth of G.oxydans WSH-003. Besides, no obvious degradation of2-KLG could be detected when G.oxydans WSH-003was grown with2-KLG for120h. In order to construct G. oxydansengineered strains, a E. coli-G. oxydans shuttle vector is essential. The par-rep gene from theplasmid pGOX3of G. oxydans621H was ligated into pUC18, resulting in the shuttle vectorpGUC, which has a high conversion efficiency and stability.(4) Construction of G. oxydans engineered strain for the direct production of2-KLG andthe modification by connecting peptide engineering and cofactor engineeringDifferent combinations of five SDH/SNDH and two SNDH from K. vulgare WSH-001were introduced into G. oxydans WSH-003, an industrial strain used for the conversion ofD-sorbitol to L-sorbose. The optimum combination produced4.9g·L–1of2-KLG. In addition,10different linker peptides were used for the fusion expression of SDH/SNDH and SNDH inG. oxydans. The best recombinant strain produced31.2g·L–1of2-KLG after168h. After theoptimization of yeast extract concentration, the production of2-KLG reached32.4g·L–1.Furthermore, overexpression of pqqA and pqqABCDE gene clusters under the control of tufBpromoter enhanced PQQ concentration by126.1%and273.6%, respectively, compared withthat noted in the wild-type strain. Overexpression of PQQ biosynthesis gene(s) significantlyenhanced cell growth, and the2-KLG production by G.oxydans/pGUC-k0203-GS-k0095-pqqA and G. oxydans/pGUC-k0203-GS-k0095-pqqABCDEreached35.1g·L–1and39.2g·L–1, which was8.3%and21.0%higher than that by G.oxydans/pGUC-k0203-GS-k0095, respectively.(5) Efficient optimization of G. oxydans based on trimeric protein CutA for the directproduction of2-KLGThe codon optimization was conducted to further improve the expression of SDH andSNDH in G. oxydans, the production of2-KLG was33.2g·L–1after the codon optimization.In order to verify whether the expression of cutA could take effect in G. oxydans, after thecodon optimization of cutA, G. oxydans strain harboring pGUC-tufB-cutA was constructed.The growth curve of G. oxydans/pGUC-tufB-cutA and the wild strain were determined, whichindicated that CutA was correctly expressed in G. oxydans, and the heat resistance of G.oxydans was improved. Furthermore, based on the adaptor protein SH3and its ligand SH3lig, the codon-optimized cutA and sdh-GGGGS-sndh were expressed in G. oxydans. Theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced40.3g·L–1of2-KLG. In order to investigate the fermentation performance at different temperatures, theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA was fermented at30?,35?and37?, respectively. G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA grew better at35?and37?than the control strain. The production of2-KLG of G.oxydans/pGUC-tufB-k0203-GS-k0095-cutA were higher than the control at differenttemperatures, which revealed that the expression of CutA improved the catalytic efficiency ofthe dehydrogenases. However, the strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced less2-KLG at35?and37?than30?. Utimately, pqqABCDE gene clusterswere overexpressed and the production of2-KLG reached to42.6g·L–1.