| Microbial cell factories can produce a variety of chemicals from abundant biomass.Their production capacity,such as titer,yield and productivity,often depend on the intracellular carbon flow,direction and rate.Therefore,the carbon flux regulation is critical to construct efficient microbial cell factory.In the process of developing high effective microbial cell factories,there are many problems,such as how to improve the precision of carbon flux regulation,how to balance the carbon flux distribution between cell growth and product synthesis,how to boost the intelligence,timeliness and universality of the regulation tools.From the perspective of synthetic biology and systematic metabolic engineering,strategies such as integration of in vitro modular optimization and CRISPRi,dynamic consolidated bioprocessing and protease-based gene circuits were put forward using E.coli as the research host,which effectively improved the microbial fermentation indexes of organic acids including malate,xylonate and shikimate.The main results are as follows:1.E.coli B0013 was used as the starting strain,and the optimal catalytic ratio of five enzymes in non-cyclic oxidation glyoxylate pathway was obtained through in vitro re-construction and modular optimization for malate production.A chassis strain E.coli B0044 was constructed by gene combination knockout to accumulate 5.30 g L-1 malate.The optimal strain E.coli B0047 was further obtained by screening and assembling guide RNAs with different suppression strengths for targeted pathway enzymes and using in vivo multiplexed CRISPRi tuning to rationally regulate the proportion of pathway enzymes.Finally,36 g L-1 malate with a yield of 0.74 mol mol-1 glucose could be achieved in E.coli B0047 by fed-batch fermentation.2.To obtain E.coli with high xylan utilization efficiency,xylan-degrading enzymes and xylose transporter proteins from different sources were screened and further modular optimized.Furthermore,by integrating and optimizing QS system and Cre recombinant enzyme system,a novel cell-density-dependent dynamic regulation switch was constructed,showing good tunability and portability.Finally,by integrating the above two systems,a novel concept termed dynamic consolidated bioprocessing technology was put forward.On this basis,engineered E.coli could produce 16.8 g L-1 xylonate,11.8 g L-1 xylitol and 3.2 g L-1 shikimate from 20 g L-1 xylan.3.Two basic protein regulatory units were constructed by combining viral proteases and protein degradation signals.The maximum regulatory fold of the two regulatory units exceeded 20,showing good tunability and specificity.On this basis,three programmable biomolecular switches were constructed.The first tool termed protease-based dynamic regulation circuit?pbDRC?,with switch time of 7-10 h is designed with GPP controlling the expression of target protein and SPP controlling the expression of protease.At the same time,a second molecular biological switch protease-based inverter?pbI?is designed either by expressing two proteases?TEVp,TVMVp?with orthogonal cutting activity or employing cascade degradation of proteases.The third molecular biological switch is a protease-based oscillator?pbO?with cascade degradation of three orthogonal proteases?TEVp,TVMVp,SuMMVp?.For the first time,an pbO with an oscillation period of 90 min was constructed at protein level.4.The effect of pbDRC on the production of shikimate in minimal medium without adding aromatic amino acids and inducers was studied.The optimal strain E.coli DS7 could achieve 2.14 g L-1 shikimate in shaker flask.Compared with that of shaker flasks,a 5.9-fold higher shikimate titer(12.6 g L-1,yield of 0.19 g g-1 glucose)was achieved in fed-batch fermentation.When pbDRC was tested in other E.coli variants,all the pbDRC-containing strains achieved increased shikimate titer ranging from 7-to 43-fold compared with that of the control strains,respectively.Genomic integration of pbDRC was also attempted in E.coli MG1655,all strains with pbDRC exhibited a 4-to 9-fold titer without acetate accumulation in comparison with the control strains.5.The effects of pbI on relieving carbon metabolism repression were studied with PTS system as the target.The pbI can be used to block glucose utilization after the biomass reaches a certain density using pbI.The optimal induction time is 6 h.On this basis,when the sugar ratio was controlled at 2(20 g L-1 glucose and 10 g L-1 xylose),a 1.79-fold D-xylonate titer increase(4.25 g L-1)was achieved over that of the control strain(2.37 g L-1).6.The effect of pbO on the production of xylonate by regulating the rate of dual-enzyme cascade reaction was studied.The titer of xylonate in strain containing pbO was 2.02 times higher than that of the control strain.Meanwhile,intracellular pH homeostasis was achieved in strain containing pbO even the extracellular pH was 4.This further led to the observed increase in cell viability by more than two orders of magnitude.After fermentation conditions optimization,13.1 g L-1 xylonate could be obtained in shaker flask in TB medium at 37°C.Lastly,a titer of 199.4 g L-1,with a productivity of 7.12 g L-1 h-1 was achieved in fed-batch fermentation. |
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