Temporo-spatial Metabolic Regulation Of Escherichia Coli For Poduction Of Fine Chemicals

Escherichia coli cell factories can produce a variety of chemicals from renewable resources.The efficiency of cell factories can significantly affect the economy of industrial bioprocessing.Thus,the major challenge faced by industrial biotechnology is to develop a highperformance microbial cell factory.To achieve this goal,from the perspective of microbial physiology,strategies such as periplasmic engineering,dynamic control of cell growth,lifespan engineering,and apoptosis engineering were put forward using Escherichia coli as a model system.The performance of microbial cell factory was effectively enhanced by limiting crosstalk between the engineered pathways and the cellular milieu,balancing cell growth and product formation,and regulating the cell senescence and apoptosis.The main results are as follows:1.Enhancement of malate production through engineering periplasmic.Firstly,E.coli ATCC8739 was used as the parent strain,and the phosphoenolpyruvate(PEP)pool was constructed using a multigene combination knockout strategy.Then,by constructing a cytoplasmic r TCA pathway,the carbon flux from PEP was redirected to malate.Next,the Pel B periplasmic localization signal was used for targeting the r TCA pathway to the periplasm,which led to a 100% increase in malate production to 18.8 m M.Finally,the dual metabolic engineering regulation was adopted to enhance malate production by balancing the cytoplasmic-and periplasmic pathways.The malate titer was further enhanced to 193 m M,along with a yield of 0.53 mol/mol,by p H-stat fed-batch culture.2.Dynamic balancing of cell growth and butyrate biosynthesis.Firstly,by literature mining,the butyrate biosynthesis was designed and constructed,and the chassis for butyrate product was built by enhancing the acetyl-Co A supplement.Then,three dynamic devices—a turn-on switch,a turn-off switch,and a recombinase-based inverter(RBI)—were constructed based on Bxb1 recombinase.Next,by activating butyrate biosynthesis via a turn-on switch,the butyrate production was up to 6.6 g/L,and by reducing cell growth via a turn-off switch,the butyrate production was up to 10 g/L.Finally,an RBI was adopted for the dynamic dual-controlling of the distribution of acetyl-Co A between cell growth and butyrate biosynthesis.The final butyrate titer was increased to 34 g/L,with a productivity of 0.405 g/L/h.3.Improvement of butyrate production through engineering chronological lifespan.Firstly,using genetic engineering,it was found that E.coli chronological lifespan was controlled by three genes: rss B,rpo S,and ubi G.Then,by introducing rss B,rpo S,and ubi G into the butyrateproducing strain,the chronological lifespan was engineering in strain BUT-5,which led to a 40% increase in butyrate titer to 10.5 g/L.Next,a multi-output recombinase-based state machine was constructed using site-specific DNA recombinases and Ssr A-tag mediated protein degradation system.Finally,the fate of butyrate-producing BUT-6 could be divided into four modes by linking the four outputs of the multi-output recombinase-based state machine,which enhancing the butyrate production up to 29.8 g/L,with a productivity of 0.405 g/L/h.4.Increasement of the poly(lactate-co-3-hydroxybutyrate)accumulation by engineering replicative lifespan.Firstly,based on the phenotype and genetic analysis,a key target gene(csr A)for controlling the replicative lifespan of E.coli was identified.Then,by deleting the csr A in poly(lactate-co-3-hydroxybutyrate)-producing strain PLH-3,the replicative lifespan was shortened to enhance the poly(lactate-co-3-hydroxybutyrate)content by 33.3%.Next,a twooutput recombinase-based state machine was constructed using site-specific DNA recombinases and recombination directionality factor.Finally,the fate of poly(lactate-co-3-hydroxybutyrate)-producing strain PLH-4 was divided into two modes by linking the two outputs of the two-output recombinase-based state machine,which enlarging cells for more poly(lactate-co-3-hydroxybutyrate)accumulation as well as regulating the intracellular PLH distribution for improving cell growth.5.Enhancement of L-lysine production by engineering cell apoptosis.Firstly,it was found that the cell apoptosis and cell death in E.coli were accelerated by increasing intracellular reactive oxygen species under high concentrations of L-lysine.Then,based on the phenotype and genetic analysis,the key target genes(hns and arc A)for delaying the cell apoptosis and cell death of E.coli were identified.Next,according to the second codon distribution of coding sequence in E.coli whole genome,the second codon engineering was proposed and testified.Finally,the cell apoptosis and cell death were regulated by engineering second codon to enhance the L-lysine titer up to 198.3 g/L in a 7.5 L fermenter.