Analysis Of Key Physiologic And Regulatory Mechanism For Accumulation Of Pyruvic Acid In Candida Glabrata

Pyruvic acid,an important organic acid,is a key metabolite in the central metabolism of cells,which has been widely used in the food,agriculture,pharmaceutical,and other fields.In recent years,the production of pyruvic acid by microbial fermentation has attracted close attention of scholars at home and abroad.Among many microorganisms used to produce pyruvic acid,Candida glabrata is the most promising strain due to its high glucose tolerance and high acid resistance.However,the industrial utilization of this strain is affected by unclear accumulation mechanism of by-products,complex regulation of intracellular central compounds and high dissolved oxygen dependence.In this work,C.glabrata CCTCC M202019 was used as the initial strain for improving the accumulation efficiency of pyruvic acid.This aim can be realized by a combination of biochemical engineering and metabolic engineering.In this way,some challenges can be solved at three aspects:identification of main by-products and its synthesis pathways,regulation and modification of the central metabolic network of C.glabrata to enhance the metabolic flux of pyruvic acid synthesis,and improving the adaptability of the pyruvic acid fermentation process to the hypoxic environment.The main results were summarized as follows:?1?High-throughput screening of C.glabrata mutant strains with distinct differences in pyruvic acid fermentation process provides the basis for the identification of by-products.A high-throughput screening method,which combined ARTP random mutagenesis and ferric nitrate multi-well plate detection,was established for rapidly obtaining strains with distinct differences in the pyruvic acid fermentation process.The screening throughput and accuracy of this method were further improved by combining with Qpix 420 automatic pick-up instrument and automatic pipetting workstation.Nine strains of pyruvic acid over-produced strains were obtained from 30000 mutants through several rounds of high throughput screening in the preliminary screening.Through rescreening experiment in shake flask,we obtained a strain C.glabrata H6 with a 32.3%increase in pyruvic acid production and a pyruvic acid producing strain C.glabrata 4-C10 with spontaneously coagulating its fermentation broth.The availability of these mutant strains with distinct differences in pyruvic acid fermentation process provides the basis for further identification of by-products in C.glabrata pyruvic acid fermentation process.?2?Identification of the main by-products and its synthetic pathways in the production of pyruvic acid by C.glabrata.By separating and purifying the fermentation broth of C.glabrata 4-C10 mutant strain and the initial strain,combining with acid hydrolysis,spectral analysis,and monomer composition analysis,we found that polysaccharide is the main by-products in the production of pyruvic acid by C.glabrata.Then,the transcriptional level analysis of genes in the polysaccharides synthesis pathway of C.glabrata revealed that the CAGL0H02695g and CAGL0K10626g genes are important genes affecting the accumulation of extracellular polysaccharides.Knockout of CAGL0H02695g and CAGL0K10626g in the initial strain reduced the accumulation of by-product polysaccharides by 15.6%,while pyruvic acid production of C.glabrata increased by 11.8%.The experimental results also showed that knocking out the important genes seriously affects the growth of the cells.Therefore,a strategy to completely block the synthesis of polysaccharides to improve pyruvic acid accumulation does not seem feasible.However,it seems to be an effective strategy to further improve pyruvic acid accumulation by regulating the central compounds closely related to polysaccharide synthesis to coordinate strain growth and polysaccharide synthesis.?3?Regulation of pyruvic acid subcellular distribution during C.glabrata fermentation to enhance the extracellular accumulation of pyruvic acid.Some reports have shown that the accumulation of polysaccharide is closely related to the subcellular distribution of intracellular pyruvic acid and ATP.Carrier engineering strategy was used to study the effects of the subcellular distribution of pyruvic acid on the central carbon metabolic pathway and the pyruvic acid extracellular accumulation of C.glabrata.Enhancing the entry of pyruvic acid into the mitochondria can improve the central carbon metabolism pathway and pyruvic acid accumulation rate of C.glabrata.In addition,comparing the intracellular pyruvic acid content of engineered and control strains showed that higher intracellular pyruvic acid levels were not conducive to improving pyruvic acid production of C.glabrata.Plasma membrane expression of mitochondrial pyruvate carrier reduced the intracellular pyruvic acid content of C.glabrata,which enhanced pyruvic acid titer to 29.0 g/L in shake flask.Moreover,the novel plasma membrane localization detection method combining flow cytometry and sortase mediated ligation reactions was established.?4?Constructing ATP futile cycle system to alleviate the feedback inhibition of ATP on the synthesis pathway of pyruvic acid.Some reported have shown that high intracellular ATP levels are not only beneficial to the efficient synthesis of by-products such as polysaccharides,but also have serious feedback inhibition on the glycolytic pathway.Metabolic engineering strategy was used to construct an ATP futile cycle system?ATP-FCS?that efficiently decreased the intracellular ATP level in C.glabrata.Some optimization strategies for ATP-FCS were also adopted to further improve the production of pyruvic acid in C.glabrata.Based on the physiological characteristics of C.glabrata,two additional enzymatic activities,pyruvate carboxylase?PYC2p?and phosphoenolpyruvate carboxykinase?PCK1p?,were introduced into the cytoplasm of C.glabrata to construct an ATP-FCS.ATP-FCS was capable of decreasing the intracellular ATP and ROS level in C.glabrata,which increased the extracellular accumulation of pyruvic acid by 49.7%.In addition,some optimization strategies for ATP-FCS were adopted to further maximize the production of pyruvic acid in C.glabrata.Finally,40.2 g/L of pyruvic acid were obtained in 500-mL shake flask.?5?Engineering hypoxia inducible factor-1 to alleviate the dependence of the pyruvic acid fermentation process of C.glabrata on high dissolved oxygen.High extracellular dissolved oxygen accelerates the entry of intracellular pyruvate into the TCA cycle and oxidative phosphorylation pathway,resulting in the oxidative decomposition of pyruvate into CO₂,while producing large amounts of ATP.The effect of HIF-1 on pyruvic acid fermentation of C.glabrata under hypoxic conditions was studied to improve the accumulation of pyruvic acid under hypoxic conditions by metabolic engineering strategies.Based on literature mining and database analysis,it was found that the promoter region of most genes related to glucose transport and glycolytic pathways in C.glabrata has A/GCGTC motif specifically recognized by HIF-1.By introducing HIF-1 into C.glabrata,the transcriptional levels of some genes in the glucose transport and glycolysis pathways under hypoxic conditions were improved.More importantly,the pyruvic acid production of C.glabrata was increased by 32.4%under hypoxic conditions.Optimization of dissolved oxygen control conditions and knockout of proline hydroxylase in C.glabrata were further carried out to improve the stability of HIF-1,and the pyruvic acid titer of C.glabrata was further improved under hypoxic conditions.Finally,the pyruvic acid production of the strain reached 53.1 g/L when the dissolved oxygen level of the strain was 10%on the fermenter.