Research On The Synthesis Of D-allose With Engineered Saccharomyces Cerevisiae Via CRISPR/Cas9

Recently,D-Allose has attracted more and more attention due to its anti-obesity,antioxidant,anti-hypertensive,anti-tumor and anti-cancer.At present,researches on the production of D-allose by Izumoring enzymatic method are extensive.It is a better way to produce value-added D-allose from D-fructose which need D-psicose 3-epimerase(DPE)and L-rhamnose isomerase(LRI).Saccharomyces cerevisiae has been recognized as a GRAS(generally recognized as safe)by the US FDA(Food and Drug Administration).It has the advantages of safety and nonpathogenicity,clear genetic background,simple cultivation and rapid reproduction.Therefore,it is worthy to produce D-allose in S.cerevisiae.However,it is necessary to delete hxk2 gene encoding hexokinase isoenzyme 2,which can reduce the D-fructose consumption rate in S.cerevisiae.The specific researches and results are summarized as follows:(?)Construct efficient CRISPR/Cas9 gene editing system in S.cerevisiae BY4741.We integrated Cas9 gen expressed by the constitutive promoter PPGK1 into rDNA site,generated strain CS1.Then gRNA-?ade2 was ligated with plasmid p RS426,generated p RS426-gRNA-? ade2.The strain CS-2 was obtained after transforming plasmid p RS426-gRNA-? ade2 into CS1 and donor-tll2,which resulted in tll2 expression cassette was integrated into ade2 site.Meanwhile,the single plasmid p YES2-CG-?ade2 was constructed,which contains Cas9 and g Block targeting ade2.We also achieved that tll2 expression cassette was integrated into ade2 site by transforming p YES2-CG-?ade2 and donor-tll2 into BY4741,generated strain CS-3.In strain CS-3,plasmid can be lost by cultivating in YPD,which means that we can realize seamless DNA editing.The results show edit efficiency of rDNA-integrated type Cas9 was 70%,but edit efficiency was 100% via single Cas9/gRNA system.It demonstrates single Cas9/gRNA system has a better editing effect,and this system will be used in subsequent gene editing.(?)We constructed low-consumption-rate host BY4741-? hxk2 by transforming plasmid p YES2-CG-?hxk2 which targeted hxk2 gene and donor-hxk2.The strain BY4741-? hxk2 in which 44 A was replaced by C,45 C was deleted and 46 A shifted,which resulted in frame-shift mutation and premature stop codon.Then,the metabolization rate of D-fructose in mutated BY4741-?hxk2 was analyzed.The results show that the OD600 of BY4741-?hxk2 was 8.65 after 22 h fermentation,which was 6.40% higher than BY4741.Furthermore,the fructose consumption rate in BY4741-?hxk2 during 14 h fermentation was 3.35 mg?h-1 which decreased by 6.42% compared with wild strain BY4741.To conclude,compared with the wild strain,the mutant of the hxk2 gene in BY4741 can not only show growth dominance but also have low-fructose consumption rate.The results we got in this article display the advantage and the potential of BY4741-?hxk2 as the host to synthesize D-allose.(?)We constructed strain Y1 in which tandem expression cassette of dpe gene from Dorea sp.CAG317 and lri gene form Clostridium stercorarium was integrated in to hxk2 site.Then strain Y1 was cultivated in YPF and the product was analyzed via HPLC.After HPLC detection,a characteristic peak with the same peak time as the labeled product D-allose was obtained.The characteristic peak was initially determined to be D-allose which content was 1.69 g/L.