Multi-omics Analysis Of Glucoamylase Producing Aspergillus Niger Strains And The Exploration Of NADPH Cofactor Engineering

With GRAS(Generally Recognized as Safe)status,Aspergillus niger is widely used as a vital cell factory for the production of various enzyme preparations as its excellent ability of protein expression and secretion.Systematic comprehension of its metabolic regulation mechanism during the fermentation process is of considerable significance to improve the performance of this versatile cell factory through metabolic engineering.As previous studies reported that reduced sporulation favors protein secretion in A.niger,in this study we conducted a comparative genomic analysis of the non-sporulating industrially exploited A.niger strain LDM3 in China and the reference protein secretion strain CBS 513.88 to predict the key genes that might define the genetic basis of LDM3's high protein producing potential in silico.After assembly,LDM3 exhibits large chromosomal rearrangements in comparison to CBS 513.88.An alignment of the two genome sequences revealed that the majority of the 457 ORFs uniquely present in LDM3 possessed predicted functions in redox pathways,protein transport,and protein modification processes.In addition,bioinformatic analyses revealed the presence of 656 ORFs in LDM3 with non-synonymous mutations encoding for proteins related to protein translation,protein modification,protein secretion,metabolism and energy production.Based on available literature and co-expression network analysis,tupA was proposed as the key factor involved in asexual sporulation of A.niger.By knockout experiments,we showed that the ?tupA mutant displayed reduced sporulation(35%)accompanied by higher total protein secretion(65%)compared to its parental strain.Oxygen limitation is a profitable strategy for industrial glucoamylase(GlaA)overproduction by A.niger.To deepen the understanding of how transcriptional network regulated to the oxygen limitation during the industrial glucoamylase bioproduction,time-course transcriptome samples from the fed-batch cultures of a high-yield GlaA producing A.niger strain DS03043 were sequenced.Gene expression pattern analysis of 515 identified differentially expressed genes(DEGs)suggested diverse expression profiles when came into the oxygen limitation.Accompanied by the reduced biomass growth,biosynthesis of cell protein was weakened,but fatty acid catabolism pathways,GlaA biosynthesis,and provision of precursor amino acids were notably activated.These proofs confirm that cellular resource,such as energy substrates and precursors metabolites,were allocated to the highly efficient GlaA accumulation under the low energy supply condition.Moreover,the defined sterol-regulatory element-binding proteins(SREBP)transcription factor SrbB was consistently up-regulated,indicating an essential role of SrbB in the transcriptional adaption to the hypoxia.In light of our previous multi-omics analysis of glucoamylase(GlaA)overproduction process,fermentative production of enzymes could be limited by the low availability of NADPH in A.niger.Given the prediction of GSMM and available literature,nine NADPH regeneration systems were identified and tested in the lab established A.niger strain AB4.1(only carrying one copy of glucoamylase encoding gene glaA)to observe the effect of cofactor engineering on protein formation.To clarify the correlation between NADPH level and target protein synthesis,it was found that albeit intracellular NADPH supply was slightly improved after genetic perturbation in AB4.1,no significant alteration was observed on protein biosynthesis.Hence,NADPH supply could not be the bottleneck for protein production because of the low NADPH requirement in AB4.1.This study also focused on whether the oxidization of NADPH inhibits protein biosynthesis through the characterization of NADPH oxidase regulator RiaA.We observed that the overexpression of riaA did not severely hinder the accumulation of NADPH,but strikingly inhibited the expression of glaA.It could be concluded that RiaA plays a complex negative regulatory role on enzyme production from the perspective of cofactor regulation and the inhibition on protein translation and secretion.In vitro CRISPR/Cas9 system is able to trigger the transient gene editing accompanied by a weakened off-target efficiency.This thesis adopted the optimized in vitro A.niger CRISPR/Cas9 platform to efficiently construct the uridine auxotrophic mutant of a high-yield GlaA producing strain A.niger B36,providing an excellent platform for further molecular genetic engineering under a high protein secretion background.The classic Design-Build-Test-Learn(DBTL)cycle of metabolic engineering is increasingly leveraged to advance rational strain development.To optimize the protein overproduction of this smart cell factory,we reported on the implementation of DBTL cycles to identify and prioritize the most effective co-factor engineering strategies from the identical seven NADPH regeneration systems as above using B36 as host.It was determined that the enzyme-producing ability is promoted by the optimized NADPH supply only in the strain with high enzyme capacity,which inferred that the newly generated NADPH contributed more to the production of GlaA in high-producing strain other than cell growth as in the low-producing strain.To clarify the metabolic adaptation caused by the NADPH perturbation,three isolates,viz.gsdA(glucose 6-phosphate dehydrogenase.G6PDH),gndA(6-phosphogluconate dehydrogenase,6PGDH)and maeA(NADP-dependent malic enzyme.NADP-ME),out of the seven candidates were selected for further characterization through maltose-limited chemostat cultures,which displayed significant regulatory effect on intracellular NADPH level.Results showed,overexpression of NADP-ME or 6PGDH progressively enhanced the intracellular NADPH pool by 45%and 66%,respectively,while overproduction of G6PDH only showed wild-type level,which may implicate the superior roles of NADP-ME and 6PGDH as major NADPH generators in A.niger.Interestingly,G6PDH and 6PGDH performed reversely on protein production,as overproduction of G6PDH resulted in a 40%lower of protein capacity compared to the control,while it was elevated by 60%and 30%via the regulation of 6PGDH and NADP-ME separately.Besides,overproduction of NADP-ME aroused the strongest rearrangement of the metabolic flux distribution compared to engineer the pentose phosphate pathway(PPP),which improved the flux through the glycolysis and the TCA cycle,ensuring the efficient provision of the precursor metabolites for amino acid formation.At the first time,the cofactor engineering in A.niger opens a new avenue to boost protein formation in this fungal factory,of which 6GPDH acts as an optimized target.Such a cofactor regulation strategy could also be transferable to guide the rational design of other microbial cell factories.In sum,comparative genome analysis uncovered several hundred unique and mutated genes in LDM3,some of which might be associated with its aconidial phenotype and its high glucoamylase secretion capacities.Gene function experiments revealed a crucial gene tupA associated with sporulation and protein secretion.The transcriptome analysis initially depicted the global transcriptome response of the cellular adaption mechanisms to oxygen-limited cultivations.Determined by previous multi-omics analysis,NADPH is regarded as one of the main limiting factors of enzyme-producing cell factories.Systems biology studies confirmed for the first time in A.niger that by enhancing specific NADPH-generated pathways,in particular 6PGDH and NADP-ME,could progressively facilitate the intracellular NADPH supply,thus promote the protein capacity in this outstanding cell factory.