Efficient Bio-synthesis Of Glycolic Acid

Glycolic acid（Glycolate）is an importantα-hydroxy carboxylic acid which is easily degraded and absorbed and it has a good application prospect in high-grade cosmetics,food processing,pharmaceutical production and polymer degradable materials.But the traditional chemical synthesis method has some problems,such as environmental pollution and non-renewable raw materials.Therefore,total biosynthesis,as an environmentally friendly and easily available raw material,has attracted more and more attention in recent years.In preliminary study,the glycolic acid synthesis pathway was successfully constructed in Escherichia coli and glycolate was synthesized using glucose as substrate.However,the high fermentation cost and low production efficiency of whole-biological synthesis of glycolate limit subsequent production applications.To solve those challenges,the following three aspects of research were conducted:（1）A high-throughput screening platform was used to obtain engineered strains with stable yield phenotypes.In preliminary study,the glycolate production of Mgly71 was found to decline during continuous fermentation process.In order to obtain an engineered strain with stable production of glycolate,a high-throughput screening method established based on the glycolate biosensor p GBS-P_ffs-sfgfp was adopted.The glycolate production of the initial strain was 1.59 g·L^-1,and the resulting strain Mgly72 produced 2.3 g·L^-1 glycolate,which was 1.45 times than that of Mgly71 in the same medium.（2）Two samples including the fermentation samples of Mgly72 and Mgly71 were subjected to transcriptomics based on RNA-Seq technology.The results showed that the significant up-regulation of aconitate hydratase（acn A）in Mgly72 promoted the carbon source flow to glycolate synthesis.The significant down-regulation of hydroxypyruvate isomerase（hyi）,tartronate-semialdehyde synthase（gcl）reduced the depletion of glycolate to recycle the carbon source for glycolate production.In addition,the expression level of NADPH synthesis-related genes was upregulated in Mgly72.Overexpression of the pnt AB gene provides more NADPH for glycolate synthesis,which increased the glycolate production of Mgly72-pnt AB to 3 g·L^-1.Compared with Mgly71,about 40 genes with significant changes in Mgly72 were found to be related to amino acid metabolism.Based on this,the relationship between amino acid metabolism and cell growth and glycolic acid synthesis in Mgly72 strain was analyzed.It was found that the expression levels of glutamate and aspartate synthesis genes were significantly increased.The content of free amino acids in the improved fermentation medium was measured,and key amino acids such as glutamate and aspartate were found to be high,which may be the reason for the high glycolic acid yield of Mgly72 strain in the improved fermentation medium.（3）Based on the M9 fermentation medium,statistical methods were empolyed to optimize the fermentation medium for glycolate production which include the analysis of the influencing complete randomalized design,the Plackett-Burman experimental design,the path of steepest ascent methods and the response surface method and experimental design.The results of complete randomalized experiment showed that the eight factors affecting the glycolate production were corn cob hydrolysis,corn steep liquor,ammonia,Mg²⁺,Ca²⁺,Fe²⁺,Mn²⁺,Zn²⁺.Those factors were then used for further optimization.The Plackett-Burman experiment,was designed using the eight factors determined in the complete randomalized experiment and two main components of the M9fermentation medium were used.Significant positive effects were observed for X1（corn cob hydrolysate）,X7（Na₂HPO₄）,X9（NH₃·H₂O）and X10（Corn steep liquor）.The central point of the central composite design as determined by the steepest ascent experiment,consisted of the following concentrations:corn cob hydrolysate-(12 g·L^-1),NH₃·H₂O-(0.8 g·L^-1),Na₂HPO₄-(22.4g·L^-1),Corn steep liquor-(19.48 g·L^-1).The Design Expert 11 software was empolyed for the central composite design,with the value of glycolate production being used as response value.Response surface methodology was utilized in the final stages to determine the optimal levels for each factor.Thus,the optimized concentrations of the fermentation medium components are as follows:13 g·L^-1corn cob hydrolysate,1.18 g·L^-1 NH₃·H₂O,21.35 g·L^-1 Corn steep liquor,25.43 g·L^-1 Na₂HPO₄.The production of glycolate by Mgly72-pnt AB was carried out under the optimum conditions.The production of glycolate increased from 3 g·L^-1 to 9.88 g·L^-1,which was 2.3-fold higher than the M9fermentation medium.Through increasing the mixing speed and ventilation volume,Mgly72-pnt AB achieved a titer of 46.2 g·L^-1 glycolate in a 5-L bioreactor,with a production efficiency of 0.57g·（L·h）^-1.