Enhanced acetate utilization for value-added chemicals production in Yarrowia lipolytica by integration of metabolic engineering and microbial electrosynthesis.

The limited supply of reducing power restricts the efficient utilization of acetate in Yarrowia lipolytica. Here, microbial electrosynthesis (MES) system, enabling direct conversion of inward electrons to NAD(P)H, was used to improve the production of fatty alcohols from acetate based on pathway engineering. First, the conversion efficiency of acetate to acetyl-CoA was reinforced by heterogenous expression of ackA-pta genes. Second, a small amount of glucose was used as cosubstrate to activate the pentose phosphate pathway and promote intracellular reducing cofactors synthesis. Third, through the employment of MES system, the final fatty alcohols production of the engineered strain YLFL-11 reached 83.8 mg/g dry cell weight (DCW), which was 6.17-fold higher than the initial production of YLFL-2 in shake flask. Furthermore, these strategies were also applied for the elevation of lupeol and betulinic acid synthesis from acetate in Y. lipolytica, demonstrating that our work provides a practical solution for cofactor supply and the assimilation of inferior carbon sources.

Coupling riboflavin de novo biosynthesis and cytochrome expression for improving extracellular electron transfer efficiency in Shewanella oneidensis.

Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c-Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine-tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, and ybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c-Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c-Cyts expression were combined, resulting in 2.34-fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.

Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering.

BACKGROUND: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. RESULTS: In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the ß-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. CONCLUSIONS: Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis.

BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear. RESULTS: In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C14 isoform. CONCLUSIONS: This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.

Gene repression via multiplex gRNA strategy in Y. lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. RESULTS: In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. CONCLUSION: Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.

Heterologous biosynthesis and manipulation of alkanes in Escherichia coli.

Biosynthesis of alkanes in microbial foundries offers a sustainable and green supplement to traditional fossil fuels. The dynamic equilibrium of fatty aldehydes, key intermediates, played a critical role in microbial alkanes production, due to the poor catalytic capability of aldehyde deformylating oxygenase (ADO). In our study, exploration of competitive pathway together with multi-modular optimization was utilized to improve fatty aldehydes balance and consequently enhance alkanes formation in Escherichia coli. Endogenous fatty alcohol formation was supposed to be competitive with alkane production, since both of the two routes consumed the same intermediate-fatty aldehyde. Nevertheless, in our case, alkanes production in E. coli was enhanced from trace amount to 58.8mg/L by the facilitation of moderate fatty alcohol biosynthesis, which was validated by deletion of endogenous aldehyde reductase (AHR), overexpression of fatty alcohol oxidase (FAO) and consequent transcriptional assay of aar, ado and adhP genes. Moreover, alkanes production was further improved to 81.8mg/L, 86.6mg/L or 101.7mg/L by manipulation of fatty acid biosynthesis, lipids degradation or electron transfer system modules, which directly referenced to fatty aldehydes dynamic pools. A titer of 1.31g/L alkanes was achieved in 2.5L fed-batch fermentation, which was the highest reported titer in E. coli. Our research has offered a reference for chemical overproduction in microbial cell factories facilitated by exploring competitive pathway.

Engineering Saccharomyces cerevisiae to produce odd chain-length fatty alcohols.

Fatty aldehydes and alcohols are valuable precursors used in the industrial manufacturing of a myriad of specialty products. Herein, we demonstrate the de novo production of odd chain-length fatty aldehydes and fatty alcohols in Saccharomyces cerevisiae by expressing a novel biosynthetic pathway involving cytosolic thioesterase, rice α-dioxygenase and endogenous aldehyde reductases. We attained production titers of ∼20 mg/l fatty aldehydes and ∼20 mg/l fatty alcohols in shake flask cultures after 48 and 60 h respectively without extensive fine-tuning of metabolic fluxes. In contrast to prior studies which relied on bi-functional fatty acyl-CoA reductase to produce even chain-length fatty alcohols, our biosynthetic route exploits α-oxidation reaction to produce odd chain-length fatty aldehyde intermediates without using NAD(P)H cofactor, thereby conserving cellular resource during the overall synthesis of odd chain-length fatty alcohols. The biosynthetic pathway presented in this study has the potential to enable sustainable and efficient synthesis of fatty acid-derived chemicals from processed biomass.

Biosynthesis of odd-chain fatty alcohols in Escherichia coli.

Engineered microbes offer the opportunity to design and implement artificial molecular pathways for renewable production of tailored chemical commodities. Targeted biosynthesis of odd-chain fatty alcohols is very challenging in microbe, due to the specificity of fatty acids synthase for two-carbon unit elongation. Here, we developed a novel strategy to directly tailor carbon number in fatty aldehydes formation step by incorporating α-dioxygenase (αDOX) from Oryza sativa (rice) into Escherichia coli αDOX oxidizes Cn fatty acids (even-chain) to form Cn-1 fatty aldehydes (odd-chain). Through combining αDOX with fatty acyl-acyl carrier protein (-ACP) thioesterase (TE) and aldehyde reductase (AHR), the medium odd-chain fatty alcohols profile (C11, C13, C15) was firstly established in E. coli. Also, medium even-chain alkanes (C12, C14) were obtained by substitution of AHR to aldehyde decarbonylase (AD). The titer of odd-chain fatty alcohols was improved from 7.4mg/L to 101.5mg/L in tube cultivation by means of fine-tuning endogenous fatty acyl-ACP TE (TesA'), αDOX, AHRs and the genes involved in fatty acids metabolism pathway. Through high cell density fed-batch fermentation, a titer of 1.95g/L odd-chain fatty alcohols was achieved, which was the highest reported titer in E. coli. Our system has greatly expanded the current microbial fatty alcohols profile that provides a new brand solution for producing complex and desired molecules in microbes.

Identification of crucial roles of transcription factor IhfA on high production of free fatty acids in Escherichia coli.

Transcription factor engineering has unique advantages in improving the performance of microbial cell factories due to the global regulation of gene transcription. Omics analyses and reverse engineering enable learning and subsequent incorporation of novel design strategies for further engineering. Here, we identify the role of the global regulator IhfA for overproduction of free fatty acids (FFAs) using CRISPRi-facilitated reverse engineering and cellular physiological characterization. From the differentially expressed genes in the ihfAL- strain, a total of 14 beneficial targets that enhance FFAs production by above 20 % are identified, which involve membrane function, oxidative stress, and others. For membrane-related genes, the engineered strains obtain lower cell surface hydrophobicity and increased average length of membrane lipid tails. For oxidative stress-related genes, the engineered strains present decreased reactive oxygen species (ROS) levels. These gene modulations enhance cellular robustness and save cellular resources, contributing to FFAs production. This study provides novel targets and strategies for engineering microbial cell factories with improved FFAs bioproduction.

Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome CRISPRi library.

Abundant renewable resource lignocellulosic biomass possesses tremendous potential for green biomanufacturing, while its efficient utilization by Yarrowia lipolytica, an attractive biochemical production host, is restricted since the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate. Given deficient understanding of inherent interactions between inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using clustered regularly interspaced short palindromic repeats interference library in Y. lipolytica, achieving tolerance to 0.35 % (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15 % (v/v) acetic acid. The tolerance mechanism might involve improvement of cell division and decrease of reactive oxygen species level. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for improving microbial tolerance to lignocellulose-derived inhibitors and revealing underlying mechanism.

Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes.

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.

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.

Recent advances in enrichment, isolation, and bio-electrochemical activity evaluation of exoelectrogenic microorganisms.

Exoelectrogenic microorganisms (EEMs) catalyzed the conversion of chemical energy to electrical energy via extracellular electron transfer (EET) mechanisms, which underlay diverse bio-electrochemical systems (BES) applications in clean energy development, environment and health monitoring, wearable/implantable devices powering, and sustainable chemicals production, thereby attracting increasing attentions from academic and industrial communities in the recent decades. However, knowledge of EEMs is still in its infancy as only ∼100 EEMs of bacteria, archaea, and eukaryotes have been identified, motivating the screening and capture of new EEMs. This review presents a systematic summarization on EEM screening technologies in terms of enrichment, isolation, and bio-electrochemical activity evaluation. We first generalize the distribution characteristics of known EEMs, which provide a basis for EEM screening. Then, we summarize EET mechanisms and the principles underlying various technological approaches to the enrichment, isolation, and bio-electrochemical activity of EEMs, in which a comprehensive analysis of the applicability, accuracy, and efficiency of each technology is reviewed. Finally, we provide a future perspective on EEM screening and bio-electrochemical activity evaluation by focusing on (i) novel EET mechanisms for developing the next-generation EEM screening technologies, and (ii) integration of meta-omics approaches and bioinformatics analyses to explore nonculturable EEMs. This review promotes the development of advanced technologies to capture new EEMs.

Type I-F CRISPR-PAIR platform for multi-mode regulation to boost extracellular electron transfer in Shewanella oneidensis.

Bio-electrochemical systems are based on extracellular electron transfer (EET), whose efficiency relates to the expression level of numerous genes. However, the lack of multi-functional tools for gene activation and repression hampers the enhancement of EET in electroactive microorganisms (EAMs). We thus develop a type I-F CRISPR/PaeCascade-RpoD-mediated activation and inhibition regulation (CRISPR-PAIR) platform in the model EAM, Shewanella oneidensis MR-1. Gene activation is achieved (3.8-fold) through fusing activator RpoD (σ70) to Cas7 when targeting the prioritized loci upstream of the transcription start site. Gene inhibition almost has no position preference when targeting the open reading frame, which makes the design of crRNAs easy and flexible. Then CRISPR-PAIR platform is applied to up-/down-regulate the expression of six endogenous genes, resulting in the improved EET efficiency. Moreover, simultaneous gene activation and inhibition are achieved in S. oneidensis MR-1. CRISPR-PAIR platform offers a programmable methodology for dual regulation, facilitating in-depth EET studies in Shewanella spp.

Transcriptome Analysis to Identify Crucial Genes for Reinforcing Flavins-Mediated Extracellular Electron Transfer in Shewanella oneidensis.

Flavins serve as the electron mediators in Shewanella oneidensis, determining the extracellular electron transfer (EET) rate. Currently, metabolic engineering of flavins biosynthetic pathway has been studied for improving EET. However, the cellular response triggered by flavins that contribute to EET remains to be elucidated. In this study, the riboflavin-overproducing strain C5 (expressing the flavins synthetic genes in plasmid PYYDT) and the PYYDT strain (harboring the empty plasmid PYYDT) in the microbial fuel cells are applied for comparative transcriptomic analyses to investigate beneficial gene targets that could improve EET. From the differentially expressed genes, we select the significantly upregulated and downregulated genes for inverse engineering in S. oneidensis. The results show that overexpression of ahpC and ccpA, and inactivation of pubA, putB, and tonB are able to improve the EET capability. Combinatorial modulation of these five genes results in the recombinant strain CM4, achieving the maximum power density of 651.78 ± 124.60 mW/m2, 1.97 folds of the parental strain. These genes modulation is speculated to reduce the ROS damage and to promote cytochrome synthesis and heme accumulation, which coherently enhance EET. Our findings facilitate in-depth understanding of the mechanism of flavins-mediated EET and provide new insights in promoting EET of S. oneidensis for electricity generation.

Non-homologous End Joining-Mediated Insertional Mutagenesis Reveals a Novel Target for Enhancing Fatty Alcohols Production in Yarrowia lipolytica.

Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in Yarrowia lipolytica. One batch of strains transformed with fatty acyl-CoA reductase gene (FAR) showed significant differences (up to 70.53-fold) in fatty alcohol production. Whole-genome sequencing of the high-yield strain demonstrated that a new target YALI0_A00913g ("A1 gene") was disrupted by NHEJ-mediated integration of partial carrier DNA, and reverse engineering of the A1 gene disruption (YlΔA1-FAR) recovered the fatty alcohol overproduction phenotype. Transcriptome analysis of YlΔA1-FAR strain revealed A1 disruption led to strengthened protein synthesis process that was confirmed by sfGFP gene expression, which may account for enhanced cell viability and improved biosynthesis of fatty alcohols. This study identified a novel target that facilitated synthesis capacity and provided new insights into unlocking biosynthetic potential for future genetic engineering in Y. lipolytica.

CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) of electroactive microorganisms (EAMs) is the dominating factor for versatile applications of bio-electrochemical systems. Shewanella oneidensis MR-1 is one of the model EAMs for the study of EET, which is associated with a variety of cellular activities. However, due to the lack of a transcriptional activation tool, regulation of multiple genes is labor-intensive and time-consuming, which hampers the advancement of improving the EET efficiency in S. oneidensis. In this study, we developed an easily operated and multifunctional regulatory tool, that is, a simultaneous clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation (CRISPRa) and interference (CRISPRi) system, for application in S. oneidensis. First, a large number of activators were screened, and RpoD (σ70) was determined as the optimal activator. Second, the effective activation range was identified to be 190-216 base upstream of the transcriptional start site. Third, up- and downregulation was achieved in concert by two orthogonal single guide RNAs targeting different positions. The activation of the cell division gene (minCDE) and repression of the cytotoxic gene (SO_3166) were concurrently implemented, increasing the power density by 2.5-fold and enhancing the degradation rate of azo dyes by 2.9-fold. The simultaneous CRISPRa and CRISPRi system enables simultaneous multiplex genetic regulation, offering the potential to further advance studies of the EET mechanism and application in S. oneidensis.

Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1.

Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5'-TTTV-3' and 5'-ATTN-3'. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.

Development of Whole Genome-Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.

Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations.

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.