| Erythritol is a four carbon polyol,which is often used as a low calorie sweetener in food production.As a GRAS strain recognized by the Food and Drug Administration of the United States,Y.lipolytica is the first choice for erythritol production.Due to aerobic and other reasons,the strain produces more heat during fermentation.However,its growth temperature is generally not more than 30°C,so it is faced with the cooling problem in summer,which leading to production stagnation,contamination,etc.,and these become important factors to push up the cost.In addition,there are many inbred lines of Y.lipolytica,which have great differences in chromosome structure and are difficult to metabolize.In order to improve the thermal tolerance and fermentation yield,the heat-resistant strain was obtained by adaptive evolution,and the key pathway genes of heat tolerance were found by transcriptome analysis.The whole genome sequencing of the wild-type strain was carried out,and the molecular operation basis was established.The heat-resistant mechanism of Y.lipolytica was revealed by molecular modification of heat-resistant key pathway genes.The transcriptional factor Ery D was used to construct a biosensor as a high-throughput screening tool responding to erythritol,and a combination of mutant library,high-throughput screening and metabolic engineering transformation was used to obtain thermotolerant and high-yield strains.The main results are as follows:?1?Through adaptive evolution,the strain BBE-18 which can tolerate 35°C was obtained,but the fermentation period was prolonged.Based on transcriptome data,the thermotolerance mechanism of BBE-18 was discussed from the perspectives of GO function,KEGG pathway,central carbon metabolism pathway,amino acid biosynthesis pathway,ergosterol and trehalose synthesis pathway.These pathways are mainly related to the metabolism of cell material and energy,and the blocking of these metabolism is the main reason for the inhibition of cell growth under heat stress.In the thiamine pathway,thiamine pyrophosphate is the cofactor of several key genes in the above-mentioned pathways,and the intracellular thiamine content directly affects the protein abundance of ATP synthase.Therefore,mining genes related to heat tolerance in thiamine pathway is conducive to solving the problem.At the same time,it was found that acid phosphatase could improve the thermotolerance of Y.lipolytica.In addition,as a foraging reaction,filamentous growth signaling pathway is beneficial to resist the lack of intracellular substance and energy.From the above three aspects,12 genes related to heat tolerance were found in this study:YALI0E32681g,YALI0E35222g,YALI0A12573g,YALI0F26521g,YALI0E00110g,YALI0F2573g,YALI0C14938g,YALI0A08800g,YALI0D01331g,YALI0E23430g,YALI0C15609g,YALI0E33803g.?2?The genome sequence of wild-type strain BBE-17 was obtained by whole genome sequencing,and severe structural variation was found by collinearity analysis,which provided information for molecular manipulation.With the help of hydroxyurea,the key non-homologous end joining gene Ku70 in BBE-17 was knocked out,which laid the foundation for the subsequent molecular operation.Using 5-fluoroorotic acid as screening marker,URA3 was knocked out to construct uracil deficient strain.Next,the 12 genes related to thermotolerance were expressed in the model strain PO1f with plasmid p YLXP'as the vector.Except YALI0C14938g,the expression of other single genes could improve the thermal tolerance of PO1f.The combination expression of YALI0E23430g,YALI0A08800g and YALI0E35222g was better,and the heat resistance effect of YALI0A08800g and YALI0E35222g combination was the most prominent.Under the condition of 3°C higher than the normal culture temperature?30°C?,the OD of thermotolerant strain was 2.18 times compared to control strain.Furthermore,the thiamine pathway was optimized,and the combination expression of YALI0E32681g,YALI0A12573g and YALI0F26521g was the best,and the OD of the stationary phase at 33°C was 3.70 times that of the control strain.The optimal scheme was expressed in wild strain BBE-17?Ku70?URA3,and the strain BBE-17T was constructed.The OD of the stationary phase at 33°C was 2.5 times compared to the control strain BBE-17.?3?Using Ery D,a transcription regulator from Brucella abortus,as a sensor,e GFP as a reporter gene was inserted into the skeleton plasmid p ET-22b?+?and transformed into E.coli BL21 to construct a biosensor indicator strain?SMB?with erythritol as effector.It was found that the functional structure of Ery D was tetramer.Under the action of erythritol,Ery D was decomposed into two dimers,which fell off the target site,exposed the transcription initiation site and started gene transcription.Extracellular thermodynamic analysis showed that Ery D had obvious affinity to DNA binding sequence?DBS?and erythritol,and its binding was mainly affected by van der Waals force or hydrogen bond.In the construction of SMB,e GFP was expressed in E.coli BL21 by p DBS,a promoter containing DBS sequence,but fluorescence could not be observed under fluorescence microscope.Based on this,in the plasmid p ET-22b?+?,Lac O and lac I were replaced by DBS and Ery D respectively.Then it was truncated from the upstream of DBS gradually.When it was cut to 104 bp,the construction was successful.The response accuracy of SMB was 250-500 m M in erythritol environment.Under the influence of 50-140 g·L-1 glucose,the response accuracy of erythritol was 5-250 m M.The successful construction of SMB lays a foundation for the subsequent high-throughput screening.?4?The mutant library of thermotolerant yeast BBE-18 was constructed by compound mutagenesis.The fermentation conditions were optimized to make the erythritol content in the fermentation broth within the SMB response range.Then,the mutant library was screened by the high-throughput method,and four heat-resistant and high-yield strains with by-products 84%lower than BBE-18 were obtained.Under the same fermentation conditions in shake flask,the yield of yli UA8 reached 103 g·L-1,which was 1.4 times higher than that of the original strain BBE-18;in a 3-L fermentor,the yield of yli UA8 reached 148 g·L-1 by fed fermentation.In order to prevent the consumption of erythritol,EYK1 and EYD1 were knocked out respectively.It was found that the effect of EYK1 knockout was not obvious,while the elimination of EYD1could effectively prevent the degradation of erythritol and the by-products disappeared.The inducible promoter p EYK1 was used to overexpress the key gene FBP of gluconeogenesis pathway to reduce the downward movement of carbon flux and promote the metabolic flux turning to pentose phosphate pathway.However,when titer of erythritol reached 10 g·L-1,OD decreased rapidly,which could not increase erythritol production.When TKL was overexpressed by a strong inducible promoter p EYD1,the metabolic flux was pulled into the pentose phosphate pathway.The cell grew well and the erythritol yield reached 206 g·L-1,which was 39%higher than that of yli UA8. |
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