Effect Of Exporters On 5-Aminolevulinic Acid Fermentation By Recombinant Escherichia Coli

5-aminolevulinic acid(ALA)is a nonprotein amino acid which is widely existing in the metabolism of organisms.ALA is a common precursor of tetrapyrrole compounds in various organisms.Because of its unique photochemical activity,there are many applications in agriculture and medicine field.Compared with chemical synthesis,the production of ALA by fermentation can greatly reduce the cost and has attracted wide attention.In the ALA production bacteria,with the increase of intracellular ALA concentration,the rate of tetrapyrrole synthesis also increased,which was not conducive to the further accumulation of ALA.The consumption of ALA can be reduced by exporting ALA out of cell and the content of ALA in the cell will be kept at a low level.Although the specific exporter of ALA has not been found yet,the structure of some small molecules such as amino acids is similar to that of ALA,and the specific exporters of these small molecules have been reported.Therefore,the main objective of this article is to explore the effects of these exporters on ALA secretion and to screen the transporters that facilitate the production of ALA.First,according to the literature,16 exporters were selected and the corresponding recombinant strains were constructed respectively.Then the effect of overexpression of different exporters on the ALA extracellular accumulation was compared in the optimized medium.The overexpression of rhtA gene increased the yield of ALA by 10.5%to 1.73 g·L-1.After that rhtA gene was knocked out by CRISPR/Cas9 gene editing technique,and the effect of different exporters on the production of ALA in a rhtA gene deletion host was further studied.The extracellular accumulation of ALA increased by 12.5%compared with the gene knockout strain when rhtA gene was overexpressed.Meanwhile rhtB,rhtC and yeeO gene overexpression could increase the extracellular ALA accumulation.Because of the poor stability of ALA in high temperature and alkaline environment,in order to determine the intracellular ALA concentration in time and accurately,a combination of liquid nitrogen and hydrochloric acid has been adopted on cell disruption.Meanwhile,these exporters which have promoted ALA extracellular accumulation were knocked out by CRISPR/Cas9 gene editing technique respectively.After constructing a series of strains with gene overexpression or deletion,this study analyzed the effects of different exporters on the export of ALA by comparisons of intracellular and extracellular ALA concentration.It was found that RhtA could significantly reduce the intracellular ALA concentration and increase the extracellular ALA concentration.In addition,overexpression of RhtB,RhtC and YeeO had little effect on both intracellular and extracellular ALA concentration.It can be concluded from the results that only RhtA is able to increase ALA production through exporting ALA out of cell efficiently.Finally,the feed fermentation in 20 L fermentor showed that E.coli W3110(DE3)-hemA-rhtA strain ALA extracellular concentration reached 13.68 g L-1,which was 29.4%higher than that of E.coli W3110(DE3)-hemA.