Cloning,Expression And Functional Analysis Of Lactic Acid Fermentation Related Genes In Aureobasidium Pullulans

Aureobasidium pullulans,as a filamentous fungus,has growth stages such as yeast-like,expanded and thick-walled spores,and possesses the ability to produce pullulan,an important organic matter.This study first discovered that A.pullulans NG has the ability to produce lactic acid.The lactic acid,as an organic acid that occupies an important position in the world,is also an indispensable industrial compound.In this study,the fermentation broth of A.pullulans NG was tested and it was first discovered that it has the ability to produce lactic acid.In recent years,the application of polylactic acid has become more common,and the demand for lactic acid has also increased.A.pullulans NG as acid-producing yeast producing lactic acid,compared with bacterial fermentation and rhizopus fermentation,may have a great role in promoting the study of lactic acid fermentation,therefore,the lactic acid fermentation of A.pullulans NG has far-reaching theory and industrial application value.Lactate dehydrogenase is a kind of oxidoreductase,its function is to catalyze the mutual transformation between pyruvic acid and lactic acid.It is a typical representative of a variety of isoenzymes,and it is of great significance in clinical diagnosis and biological evolution.The NG lactate dehydrogenase gene of A.pullulans had not been reported before,and it was confirmed by combining transcriptome-related data that whether the lactic acid fermentation-related gene of A.pullulans NG is a gene of great significance.The new LDH gene Not only can it contribute to gene pools,but it can also enrich the information system for detecting genetic evolution.A new generation of high-throughput sequencing technology?Illumina Hi-Seq?was used to perform transcriptome sequencing of A.pullulans NG grown in YPD medium,and transcriptome-related data were used to analyze and screen genes involved in lactic acid fermentation.The RT-qPCR technique was used to detect the transcription level of lactic acid fermentation-related genes in cDNA samples under the conditions of lactic acid synthesis.Based on the relevant sequence information in the transcriptome,A.pullulans NG cDNA was used as a template to amplify lactic acid fermentation-related gene comp5964.The cloning vector DH5?-pMD-ldh and the expression vector Rosetta-Pet32a-ldh were constructed and Protein LDH was finally induced by IPTG.The target band with a size of about 61 KD was visible by SDS-PAGE,which was consistent with the expected size.The activity of the recombinant protease was 11U/ml as determined by enzyme activity,indicating that the LDH gene expression product of the A.pullulans had biological activity.The optimal temperature for the recombinant lactate dehydrogenase in the enzymatic reaction is 35?,the optimum pH condition is 6.4,and the thermal stability is relatively good.The enzyme activity is affected by different metal ions,among which Mg²⁺LDH activity is promoted,while other ions(Zn²⁺,Cu²⁺,Na⁺,K+,Fe³⁺,and Ca²⁺)inhibit the activity of the enzyme.