多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

3-羟基丙酸(3-HP)是公认的高附加值化学品之一,可用于合成多种化学品,例如丙烯酰胺、丙烯酸、1,3-丙二醇以及可降解塑料.目前3-HP的生物合成过程均以糖为原料,且需求不断增长,亟需寻找不依赖耕地的替代原料.碳一资源来源广泛,是理想的生物制造原料.其中,甲醇可由煤、天然气制得,也可由CO_(2)加氢制备.多形汉逊酵母作为典型的甲基营养型酵母,能够以甲醇为唯一碳源和能源进行生长,并且能够耐受高温、高渗透压和低pH等条件,是非常优秀的工业菌株.近年来基因编辑技术的发展,大大加快了多形汉逊酵母代谢工程改造,有望构建高效细胞工厂,实现甲醇生物转化.本文以多形汉逊酵母为宿主,系统改造其细胞代谢实现甲醇高效转化为3-HP.由于汉逊酵母自身不能合成3-HP,因此需要表达来源于Chloroflexus aurantiacus的3-HP合成途径关键基因MCR,该途径以乙酰-CoA和丙二酰-CoA为前体,以还原力NADPH为辅因子.首先优化MCR表达,采用融合蛋白并结合甲醇诱导型启动子PAOX基因组整合MCR表达, 3-HP合成效率最高;其次,强化供应前体乙酰-CoA以及丙二酰-CoA,使3-HP产量提高26%;进一步改造氧化磷酸戊糖途径以增强辅因子NADPH的供应,使3-HP产量提高30%;最后,将增强前体和辅因子供应的有效策略结合,工程菌HP15相比出发菌HP07摇瓶发酵3-HP产量提高135%,达到1.45 g/L.通过摇瓶补料分批发酵, 3-HP产量达到7.10 g/L,得率达到0.14 g/g甲醇,为目前报道碳一资源合成3-HP的较高产量,表明多形汉逊酵母作为甲醇细胞工厂合成化学品具有很好的潜力.预期结合“液态阳光”CO_(2)制备甲醇技术以及甲醇生物转化过程,有望将CO_(2)制备成高附加值化学品,有助于实现碳中和.

Development of an indirect immunofluorescence assay for PCV3 antibody detection based on capsid protein

Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.

Genetic tools for metabolic engineering of Pichia pastoris

The methylotrophic yeast Pichia pastoris(also known as Komagataella phaffii)is widely used as a yeast cell factory for producing heterologous proteins.Recently,it has gained attention for its potential in producing chemicals from inexpensive feedstocks,which requires efficient genetic engineering platforms.This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P.pastoris.The topics cover promoters,terminators,plasmids,genome integration sites,and genetic editing systems,with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools.Additionally,this review highlights the prospects of multiplex genome integration,fine-tuning gene expression,and single-base editing systems.Overall,the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P.pastoris.

An improved CRISPRi system in Pichia pastoris

CRISPR interference(CRISPRi)has been developed and widely used for gene repression in various hosts.Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains.The CRISPRi system shows strong repression of eGFP,with the highest efficiency of 85%.Repression of native genes is demonstrated by targeting AOX1 promoter.AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium.Effects of gRNA expression and processing on CRISPRi efficiency is also investigated.It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression,and Csy4 cleavage shows higher repression efficiency.However,gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP.By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites,both genes can be repressed simultaneously.Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes(FAA1 and FAA2).Both genes are efficiently repressed,demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P.pastoris.

Diagnosis of COVID-19 Pneumonia via a Novel Deep Learning Architecture

COVID-19 is a contagious infection that has severe effects on the global economy and our daily life.Accurate diagnosis of COVID-19 is of importance for consultants,patients,and radiologists.In this study,we use the deep learning network AlexNet as the backbone,and enhance it with the following two aspects:1)adding batch normalization to help accelerate the training,reducing the internal covariance shift;2)replacing the fully connected layer in AlexNet with three classifiers:SNN,ELM,and RVFL.Therefore,we have three novel models from the deep COVID network(DC-Net)framework,which are named DC-Net-S,DC-Net-E,and DC-Net-R,respectively.After comparison,we find the proposed DC-Net-R achieves an average accuracy of 90.91%on a private dataset(available upon email request)comprising of 296 images while the specificity reaches 96.13%,and has the best performance among all three proposed classifiers.In addition,we show that our DC-Net-R also performs much better than other existing algorithms in the literature.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engi-neering.Ogataea polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,thermal-tolerance,and capacity to achieve high-density fermentation.However,a limited number of available promoters hinders the engineering of O.polymorpha for bio-productions.Here,we systematically characterized native promoters in O.polymorpha by both GFP fluorescence and fatty alcohol biosynthesis.Ten constitutive promoters(P_(PDH),P_(PYK),P_(FBA),P_(PGM),P_(GLK),P_(TRI),P(GPI),P_(ADH1),P_(TEF1) and P_(GCW14))were obtained with the activity range of 13%–130% of the common promoter P_(GAP)(the promoter of glyceraldehyde-3-phosphate de-hydrogenase),among which P_(PDH) and P_(GCW14) were further verified by biosynthesis of fatty alcohol.Furthermore,the inducible promoters,including ethanol-induced P_(ICL1),rhamnose-induced P_(LRA3) and P_( LRA4),and a bidirectional promoter(P_(Mal)-P_(Per))that is strongly induced by sucrose,further expanded the promoter toolbox in O.polymorpha.Finally,a series of hybrid promoters were constructed via engineering upstream activation sequence(UAS),which increased the activity of native promoter P LRA3 by 4.7–10.4 times without obvious leakage expression.Therefore,this study provided a group of constitutive,inducible,and hybrid promoters for metabolic engineering of O.polymorpha,and also a feasible strategy for rationally regulating the promoter strength.

Construction of microbial chassis for terpenoid discovery

Natural products are one of the major sources of small-molecule pharmaceutical drugs[1,2].However,discovery of novel natural products to expand the pool of potential drug candidates is becoming more and more challenging.Most of the traditional molecule discovery methods are based on direct separation of natural product from its natural hosts or homology search of biosynthetic pathways.However,there are currently two main obstacles in discovery of natural products from their native hosts:1)the difficulty in cultivation of the native hosts under laboratory conditions,2)no or not enough production of target natural products due to the silence of the biosynthetic pathway genes in the native hosts.Therefore,novel platforms are required for efficient discovery of new natural products.