A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter(pAOX1).However,it is difficult to use P.pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination(HR)efficiency and the limitation of handy selective markers,especially in the condition of multistep biosynthetic pathways.Hence,we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker(HiEE-ReSM)to facilitate cell factory in P.pastoris.Firstly,we improved the HR rates of P.pastoris through knocking out the non-homologous-end-joining gene(Δku70)and overexpressing HR-related proteins(RAD52 and RAD59),resulting in higher positive rate compared to the basal strain,achieved 97%.Then,we used the uracil biosynthetic genes PpURA3 as the reverse screening marker,which can improve the recycling efficiency of marker.Meanwhile,the HR rate is still 100%in uracil auxotrophic yeast.Specially,we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter(scFUR4)to increase the uptake of exogenous uracil from medium.Meanwhile,we explored the optimal concentration of uracil(90 mg/L)for strain growth.In the end,the HiEE-ReSM system has been applied for the inositol production(250 mg/L)derived from methanol in P.pastoris.The systems will contribute to P.pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon(C1)bio-utilization.

Enhanced biohydrogen generation from organic wastewater containing NH^(+)_(4)by phototrophic bacteria Rhodobacter sphaeroides AR-3

NH^(+)_(4)is typically an inhibitor to hydrogen production from organic wastewater by photo-bacteria.In this experiment,biohydrogen generation with wild-type anoxygenic phototrophic bacterium Rhodobacter sphaeroideswas found to be sensitive to NH^(+)_(4)due to the significant inhibition of NH^(+)_(4)to its nitrogenase.In order to avoid the inhibition of NH^(+)_(4)to biohydrogen generation by R.sphaeroides,a glutamine auxotrophic mutant R.sphaeroides AR-3 was obtained by mutagenizing with ethyl methane sulfonate.The AR-3 mutant could generate biohydrogen efficiently in the hydrogen production medium with a higher NH^(+)_(4)concentration,because the inhibition of NH^(+)_(4)to nitrogenase of AR-3 was released.Under suitable conditions,AR-3 effectively produced biohydrogen from tofu wastewater,which normally contains 50–60 mg/L NH^(+)_(4),with an average generation rate of 14.2 mL/L$h.This generation rate was increased by more than 100%compared with that from wild-type R.sphaeroides.