Highly efficient multiplex base editing:One-shot deactivation of eight genes in Shewanella oneidensis MR-1

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates.Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1.Since multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in S.oneidensis.Four approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster.Then,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,respectively.To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were screened.This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain.The maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type strain.This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.

Conductive proteins-based extracellular electron transfer of electroactive microorganisms

Electroactive microorganisms(EAMs)could utilize extracellular electron transfer(EET)pathways to exchange electrons and energy with their external surroundings.Conductive cytochrome proteins and nanowires play crucial roles in controlling electron transfer rate from cytosol to extracellular electrode.Many previous studies elucidated how the c-type cytochrome proteins and conductive nanowires are synthesized,assembled,and engineered to manipulate the EET rate,and quantified the kinetic processes of electron generation and EET.Here,we firstly overview the electron transfer pathways of EAMs and quantify the kinetic parameters that dictating intracellular electron production and EET.Secondly,we systematically review the structure,conductivity mechanisms,and engineering strategies to manipulate conductive cytochromes and nanowire in EAMs.Lastly,we outlook potential directions for future research in cytochromes and conductive nanowires for enhanced electron transfer.This article reviews the quantitative kinetics of intracellular electron production and EET,and the contribution of engineered c-type cytochromes and conductive nanowire in enhancing the EET rate,which lay the foundation for enhancing electron transfer capacity of EAMs.

CO_(2)到高附加值产物:罗尔斯通氏菌的固碳研究进展

为了实现清洁生产与碳中和的目标,研究者已采用生物固碳方法将CO_(2)转化为高附加值产物.氢氧细菌罗尔斯通氏菌由于具有化能自养能力、遗传可操作性和基因工具的可用性等特点,为自养生产的优势底盘之一.它可以利用CO_(2)作为碳源、H;作为能源自养生长,而H;则可通过直接补料、电驱动或光驱动等方法提供.罗尔斯通氏菌不仅可以天然地生产生物塑料,还能经过代谢工程改造后生产生物燃料和糖类等其他高附加值产物.本文首先介绍了罗尔斯通氏菌的碳固定和能量利用的代谢特点.然后,按照能量利用的方式,总结了该菌株直接利用H;进行气体发酵、间接利用H;(含甲酸)进行电合成和光合成的研究进展.最后,通过对比3种生产方式的优劣势,对罗尔斯通氏菌的未来研究方向进行了展望.

“共轭聚合物-产电菌”复合生物电极构建及其在微生物燃料电池中的应用

微生物燃料电池(Microbial fuel cell,MFC)作为一种生物电化学装置,在可再生能源生产和废水处理方面的巨大潜力已引起广泛关注。然而MFC面临输出功率低、欧姆内阻高以及启动时间长等问题,极大限制了其在实际工程中的应用。MFC中阳极是微生物附着的载体,对电子的产生及传递起着关键作用,开发优质的生物电极已发展成为改善MFC性能的有效途径。共轭聚合物具有成本低、电导率高、化学稳定性及生物相容性好等优点,利用共轭聚合物修饰生物电极结构,可以实现大比表面积、缩短电荷转移路径,从而实现高效生物电化学性能。同时,纳米级共轭聚合物包覆细菌,可以使细菌产生的电子有效地传递到电极。文中综述了最近报道的共轭聚合物在MFC中的应用,重点介绍了共轭聚合物修饰的MFC阳极,系统分析了共轭聚合物的优点及局限性,以及这些高效复合生物电极如何解决MFC应用中存在的低输出功率、高欧姆内阻及长启动时间等问题。

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.