| The model organism of Escherichia coli was recruited as chassis for the construction ofβ-alanine biosynthesis pathway aerobically and anaerobically.As reported,many problems existed in theβ-alanine biosynthesis pathway,which limited the production ofβ-alanine significantly.In order to solve these problems so that increasingβ-alanine production,multivariate modular strategy was applied to regulate theβ-alanine biosynthesis pathway systematically.While the production ofβ-alanine was increased gradually,the puzzle of the imbalanced metabolic flux could be detected and solved immediately,so that kept the homeostasis of the intracellular metabolic environment.Moreover,rational and semi-rational strategies were recruited respectively in the regulation of anaerobic metabolic pathway to enhance the generation of ATP,which leading to the enhancement of the growth performance and production performance of microbial cell factories.The main results are listed as follows:（1）Enhancement of the key rate-limiting step to realize the generation ofβ-alanine by Escherichia coli.As reported,a key rate-limiting step was existed in theβ-alanine biosynthesis pathway,which seriously limited the biosynthesis ofβ-alanine.The pan D gene from Corynebacterium glutamate was overexpressed on both chromosome and plasmids to enhance the key rate-limiting step so that improving the biosynthesis ofβ-alanine,therefore 4.43 g·L-1β-alanine was achieved.Moreover,yeast extract was used to provide growth factors for the growth of Escherichia coli,which not only solved the product inhibition caused by the accumulation ofβ-alanine,but also further increased the production ofβ-alanine in aerobic fermentation significantly.Finally,6.65 g·L-1β-alanine was generated in shaking flask fermentation.（2）Increasing the production ofβ-alanine in aerobic fermentation by systematical multivariate modular metabolic engineering.For the aim of channel the metabolic flux into the direction ofβ-alanine production and keep the homeostasis of the intracellular metabolic environment,the multivariate modular strategy was applied to regulate theβ-alanine biosynthesis pathway,and the completeβ-alanine biosynthesis pathway was separated into different modules,which named asβ-alanine biosynthesis module and TCA module and substrate fermentation module,according to its function and location.Experiments were carried out to balance the metabolic flux between and within each module to keep the the homeostasis of the intracellular metabolic environment.Inβ-alanine biosynthesis module,pan D gene was overexpressed to enhance the key rate-limiting step in the production ofβ-alanine.Finally,the production ofβ-alanin was up to 7.18 g·L-1.In TCA module,the synthesis of fumaric acid was enhanced and the bypass that consume fumaric acid was weakened so that increasing the accumulation of fumaric acid intracellular to provide sufficient precursor for the biosynthesis of L-aspartic acid andβ-alanine.What’s more,with the ignite of glyoxylae shunt,the generation of malic acid and oxaloacetic acid were enhanced,and the synthesis of byproduct valine was weakened,which not only enhanced the growth performance of microbial cell factories,but also improved the substrate conversion rate toβ-alanin.Finally,the production ofβ-alanin was up to 8.95g·L-1,which increased by 24.65%.In substrate fermentation module,the biosynthesis of oxaloacetic acid was enhanced to provide sufficient precursor for TCA module,and the great puzzle of pathway imbalance was solved synchronously.Finally,the production ofβ-alanin was up to 9.54 g·L-1,which increased by 6.95%.In order to further enhanceβ-alanin biosynthesis pathway,pan D gene from Tribolium castaneum was overexpressed.Ultimately,fermentation was carried out in 5 L bioreactor,and the production ofβ-alanin was up to 37.93 g·L-1,it’s 16.93 times of the start strain.It indicated that the engineered strain obtained outstanding growth performance and production performance.（3）Increasing the production ofβ-alanine in anaerobic fermentation by systematical metabolic pathway engineering.As reported,the most serious challenge in anaerobic fermentation was energy shortage.To solve this problem and increaseβ-alanine production,the multivariate modular strategy was applied to regulate theβ-alanine biosynthesis pathway,and theβ-alanine biosynthesis pathway was separated into different modules according to the location and function of each gene,and the separated modules were named asβ-alanine biosynthesis module and oxaloacetic acid biosynthesis module and substrate fermentation module.Experiments were carried out to balance the metabolic flux between and within every module and enhance the production of ATP and oxaloacetic acid,so that improving the growth performance and production performance of Escherichia coli.Inβ-alanine biosynthesis module,β-alanine biosynthesis pathway was constructed with the overexpression of heterogenous asp DH gene,and the key metabolic bottleneck was enhanced with the overexpression of heterogenous pan D genes,so that 70.87 mg·L-1β-alanine was generated.In oxaloacetic acid biosynthesis module,heterogenous pck and pyc genes were overexpressed to connect the process ofβ-alanine biosynthesis with the generation of ATP,which enable the generation of ATP along with the generation ofβ-alanine.What’s more,the regulator of cra was overexpressed to rearrange the metabolic flux and enhance the metabolic flux in pck node,channeling the metabolic flux of oxaloacetic acid biosynthesis in the direction of ATP generation,so that solving the problem of energy shortage in anaerobic fermentation and improving the generation ofβ-alanine.The regulator of arc A was knocked out to enhance the growth of Escherichia coli in anaerobic fermentation.Moreover,the gene of sbt A was overexpressed to realise the active transport of bicarbonate to enhance the activity of enzymes encoded by of ppc and pck and pyc.Finally,87.03mg·L-1β-alanine was achieved,which increased by 22.09%.In substrate fermentation module,the generation of oxaloacetic acid was improved with the enhancement of glycerol consumption process,and the production ofβ-alanine was up to 95.71 mg·L-1,which increased by 31.14%with the comparison of start strain.（4）Construction of evolutionary strains forβ-alanine production with the guide of adaptive laboratory evolution.Laboratory adaptive evolution strategy,a type of semi-rational strategy,is a supplement to rational regulation strategy.The genetic mutation library of the adapted mutant pool was established according to the sequencing results of mutant strains,which provided novel strategies for the construction of microbial cell factories.The lethal ppc deficient mutant was recruited for modified adaptive laboratory evolution,and the experiment cycle was reduced significantly with the recruition of modified adaptive laboratory evolution.With the analysis of these mutants,the mutation library of the adapted mutant pool was established,which provide the theoretics for the analyzation of the mechanism between mutant genes and mutant traits.Base on the mechanism analysed in this study,microbial cell factories could be constructed to reinforce the production of oxaloacetic acid based chemical substances in anaerobic fermentation,including the biosynthesis ofβ-alanine. |
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