Directed Regulation Of Carbon Metabolic Flux For 3-hydroxypropionic Acid Production In Klebsiella Pneumoniae

3-Hydroxypropionic acid(3-HP)is an important C3 platform compound,which can be easily converted into a series of valued chemicals or novel biodegradable materials due to the chemically active hydroxyl and carboxyl groups.Conventional chemical methods for 3-HP production were resource-consuming and environment-polluting,which constrains applications in industrial production.In contrast,bio-production is relatively inexpensive and environment-friendly,which provides a sustainable alternative for industrial production.Klebsiella pneumoniae grows rapidly,and the 3-HP synthesis pathway therein is extremely short and closely coupled with the core carbon metabolism that sustains cell growth.Therefore,K.pneumoniae is a promising host strain for production of 3-HP.Although there are various approaches to overproduce desired metabolites,direct intensification of the desired metabolic pathways is the most effective means to achieve this goal.In this study,a panel of strategies were implemented to regulate carbon flux and balance the resource into for cell growth and 3-HP synthesis in K.pneumoniae.In addition,genetic manipulation of K.pneumoniae genome could also extend the application of the strain in industrial production.The main research contents were as follows:1.To augment the expression of aldehyde dehydrogenase(ALDH),a key enzyme for 3-HP synthesis in K.pneumoniae,a cyclic AMP receptor protein(CRP)-based artificial transcription factor(ATF)was developed.The transcriptional activation domain of ATF was expected to recruit RNA polymerase(RNAP)and thereby enhance the transcription level of the puuC gene.In brief,CRP was fused to lac repressor protein(LacI)to form an ATF with the expectation that when LacI acted on the lacO positioned upstream of gene of interest,the LacI-tethered CRP would trap RNAPs and thus improve the expression of PuuC.SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and enzyme activity assay showed that the protein band of ALDH in recombinant harboring ATF was significantly stronger than that in the control group,and the enzyme activity was 23.29 U/mg,which was 45%higher than the control group without ATF.qRT-PCR analysis showed that the transcription level of puuC was 558 times that of the wild-type K.pneumoniae.The 3-HP titers of the recombinant in shake flask and 5 L bioreactor were 2.64 g/L and 35.1 g/L,respectively,indicating potential for 3-HP production.2.To regulate resource allocation during 3-HP production in K.pneumoniae,the tryptophan operon and CRISPRi were employed to engineer a metabolic toggle switch,which was recruited to regulate the expression level of growth-related glycerol kinase(encoded by glpK gene)and 3-HP synthesis-related ALDH(encoded by puuC gene)in two-stage approach.As a consequence,the metabolic flux was partitioned into cell growth and metabolite production.Specially,a suicide vector pfCB101 for gene editing was constructed,and the function of each element in the vector was determined.The dCas9 expression cassette in the CRISPRi system was successfully integrated into K.pneumoniae genome to generate the platform strain K.p-dCas9.Subsequently,five fragments were cloned into p15A-tac to obtain the recombinant vector p15A-tac-sl-tac-C-tac-tTP-sg2-trp-K(p-switch),which was used to modulate the growth and 3-HP production in K.pneumoniae.Results showed that biomass decreased by about 19%.When the growth pathway was blocked,3-HP concentration was 2.66 g/L in shake flask conditions.In a 5 L bioreactor,recombinant strain producted 47.86 g/L 3-HP,and inhibition of the growth pathway would affect the accumulation of 3-HP.3.To enable application of high-efficiency T7 expression system in non-model organisms,T7 orthogonal expression system was developed in K.pneumoniae.This orthogonal system was used to express ALDH and cell growth and 3-HP synthesis could thus be separated.First,T7 RNAP was expressed in K.pneumoniae,and the feasibility of the T7 expression system in non-model organism was investigated using fluorescent protein EGFP.Results demonstrated that the T7 expression system was compatible with the endogenous expression system in K.pneumoniae.However,the vector-dependent expression of T7 RNAP consumed substrate and thereby hindered 3-HP production.In shake flask conditions,this strain produced 1.72 g/L 3-HP and exhibited 23.26 U/mg ALDH activity.Subsequently,the T7 RNAP coding gene was integrated into K.pneumoniae genome by Red recombination system,resulting in platform strain K.p-T7.In shake flask cultivation,the strain K.p-T7(pET28a-puuC)showed improved cell growth and produced 2.44 g/L 3-HP.In a 5 L bioreactor,this strain produced>60 g/L 3-HP after 48 h,and the highest titer was 67.59 g/L.These results indicated that the engineered T7 expression system in the genome functioned well in the production of desired metabolites.4.3-HP biosynthesis in K.pneumoniae is closely associated with the core metabolism that sustains growth.To further improve 3-HP production,tandem repetitive tac promoters were harnessed to accommodate adequate RNAP and maximize the expression of endogenous puuC gene in K.pneumoniae.SDS-PAGE results demonstrated that the ALDH in the recombinant strains were normally expressed.Enzyme activity assay showed that when three repeats of tac promoter were used,the recombinant strain exhibited the highest ALDH activity(50 U/mg);Notably,qRT-PCR results showed that the transcription level of puuC was 2069 times higher than that of wild-type strain.When three tandem repetitive tac promoters were recruited to overexpress ALDH,up to 102.61 g/L 3-HP was generated.In addition,lactic acid completely vanished during late stage of fermentation,which saved the trouble of downstream separation of lactic acid from 3-HP.In order to replenish the resources required for over-expressing ALDH,the amount of nitrogen source supplemented in fermentation medium was optimized.Although 3-HP titer was not significantly improved,the accumulation was accelerated.As a result,3-HP titer reached 66.32 g/L at 24 h,and the productivity was 2.76 g/(L·h).This work provides a basis for future industrial production.