Research On 2,3-butanediol Production By Serratia Marcescens H30 And Its Metabolic Regulation

2,3-butanediol is an important biobased bulk chemical due to its extensive industrial application. It has been shown to have potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives and plasticizers, and as a carrier for pharmaceuticals. In this current thesis,2,3-butanediol production by Serratia marcescens H30 was studied by using traditional fermentation regulatory methods and modern metabolic engineering technique. The detailed work was introduced as following:1. Optimization of fermentative conditions and medium compositionsThe optimization of flask fermentation conditions and cultural medium compositions for 2,3-butanediol production by Serratia marcescens H30 was investigated. The results showed that the optimal fermentation conditions included initial pH of 7.0, process controlled to pH 6.0, cultivation at 30℃, inoculum size of 5%(v/v) and 50mL medium in 250mL flask. On the basis of the above fermentation conditions, the concentrations of medium components were optimized in shake flask fermentations by using single factor experiment, Plackett-Burman design and Response Surface methodology. And the optimal medium (g/L) (sucrose 90; yeast extract 33.36; sodium citrate 10; sodium acetate 4; MnSO4 0.1; MgSO4 0.3; NH4H2PO4 3) was obtained. It could improve 2,3-butanediol production from 15.93 g/L to 44.7g/L and shorten fermentation period from 48h to 15h. Fed-batch experiments in flask showed residual sucrose concentration of 15-30g/L favored 2,3-butanediol production.2. The experiments in 3.7L bioreactor and scale up in 50L-5000L bioreactorsThe fermentation experiments were firstly carried out in 3.7L bioreactor. Several feeding and regulatory strategies, including pulse fed batch, constant feed rate fed batch, constant residual sucrose concentration fed batch with respiratory quotient (RQ) control and pH self-control with constant residual sucrose concentration fed batch and RQ control, were compared for improving the production of 2,3-butanediol. The obtained 2,3-butanediol concentrations were 115.5g/L,117.14g/L,139.92g/L and 130.65g/L, respectively. Ultimately, a suitable control strategy which combined the RQ control with the constant residual sucrose concentration fed batch was developed. Using this strategy, the DCW (Dry Cell Weight) of 16.05g/L with the 2,3-butanediol productivity of 3.34g/L-h and the yield of 94.67%was obtained. Then we performed fermentation scale up experiments in 50L-5000L bioreactors using the above strategy. The 2,3-butanediol concentrations obtained in 50L-5000L bioreactors were over 130g/L, and in 5000L bioreactor the 2,3-butanediol concentrations of 130.2g/L was achieved.3. Construction of the swrw mutant encoding a biosurfactant synthase in S. marcescens H30Biosurfactant synthesis by S. marcescens H30 during the fermentation process for 2,3-butanediol producton results in a lot foam formation, which is harmful to the fermentation process due to microbial pollution. In addition, excessive anti-foam agent added would influence the microbial activity and emulsify fermentation broth. So we amplified and cloned the swrw gene encoding the biosurfactant synthase in S. marcescens H30. A swrw mutant by mutagenesis with the suicide vector was constructed successfully. The flask experiment results of the swrw mutant on the basis of optimized medium showed that the swrw inactivation had no effect on the growth and 2,3-butanediol production. In 3.7L bioreactor, the swrw mutant was performed fermentation experiments using constant residual sucrose concentration fed batch-RQ control strategy. The maximum 2,3-butanediol concentration of 152g/L was obtained at 57h.4. Cloning, characterization and expression of the genes involved in 2,3-butanediol pathway from S. marcescens H30Based on the genome sequence of Serratia genus, we successfully cloned the genes involved in 2,3-butanediol pathway from S. marcescens H30. The sequencing result showed the budA, budB and budR genes, encoded a-acetolactate decarboxylase (a-ALDC), a-acetolactate synthase (a-ALS) and a LysR type regulatory factor, located in one operon designated acetoin operon. While the budC gene encoded 2,3-butanediol dehydrogenase (acetoin reductase) exsited in another position of the genome. Search of the knowledge database comfirmed that this was the first report of the genes involved in 2,3-butanediol pathway from S. marcescens H30. Bioinformatics analysis showed that the strengths of the budA, budB and budC genes were 780bp,1686bp and 756bp respectively, and encoded proteins of 259,561 and 251 residues. Their molecular weights and isoelectric points were 28.96kD and 5.48,60.7kD and 5.88,27.43kD and 5.51. They were acidic proteins judged from the calculated pI values. Expression products of the genes with pET28a system exhibited comparable molecular weights using SDS-PAGE analysis.5. Regulatory mechanism of 2,3-butanediol biosynthesis by S. marcescens H30To identify the function of budR, a budR mutant by mutagensis with the suicide vector was constructed successfully. The fermentation experiments showed that the budR gene regulated the precursor (acetoin) biosynthesis of 2,3-butanediol as a positive regulatory factor. While assay of 2,3-butanediol dehydrogenase from the budR mutant showed that the budC gene encoding 2,3-butanediol dehydrogenase from S. marcescens H30 remained its catalytic activity. The results indicated that the transcription and expression of the budC gene was not regulated by the budR gene.A lacZ reporter vector pPbud-lacZ fusing the promoter of the acetoin operon and the lacZ gene from PSV-beta-gal was constructed and used to analyze the effects of pH, dissolve oxygen and organic acid on the transcriptional level of the promoter. The results showed that pH had little effect on the transcriptional level of the promoter, while dissolve oxygen and organic acid had significant effect on the transcriptional level of the promoter. The expression of the lacZ gene was raised with the dissolve oxygen lowering. For organic acids, acetic acid, lactic acid, succinic acid and citric acid could increase partially the expression of the lacZ gene, it seemed acetic aicd was the best additive of the fermentation medium.6. Construction of engineering strain for 2,3-butanediol productionIn order to decrease the accumulation of acetoin in the fermentation process for 2,3-production by S. marcescens H30, a constitutive expression vector pPbud-BDH was constructed for over-expression of 2,3-butanediol dehydrogenase (BDH) from Klebsiella pneumoniae in S. marcescens H30. Flask experiments using the engineering strain showed that over-expression of BDH in S. marcescens H30 could lower acetoin accumulation and accelerate the sucrose consumption. In 3.7L bioreactor, the engineering strain was performed fermentation experiments using constant residual sucrose concentration fed batch-RQ control strategy. The maximum 2,3-butanediol concentration of 151g/L was obtained at 42h with the 2,3-butanediol productivity of 3.59g/L-h and the yield of 94.97%.