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Abstract From both economic and environmental perspectives, ethylene glycol, the principal constituent in the degradation of PET, emerges as an optimal feedstock for microbial cell factories. Traditional methods for constructing Escherichia coli chassis cells capable of utilizing ethylene glycol as a non-sugar feedstock typically involve overexpressing the genes fucO and aldA. However, these approaches have not succeeded in enabling the exclusive use of ethylene glycol as the sole source of carbon and energy for growth. Through ultraviolet radiation-induced mutagenesis and subsequent laboratory adaptive evolution, an EG02 strain emerged from E. coli MG1655 capable of utilizing ethylene glycol as its sole carbon and energy source, demonstrating an uptake rate of 8.1 ± 1.3 mmol/gDW h. Comparative transcriptome analysis guided reverse metabolic engineering, successfully enabling four wild-type E. coli strains to metabolize ethylene glycol exclusively. This was achieved through overexpression of the gcl, hyi, glxR, and glxK genes. Notably, the engineered E. coli chassis cells efficiently metabolized the 87 mM ethylene glycol found in PET enzymatic degradation products following 72 h of fermentation. This work presents a practical solution for recycling ethylene glycol from PET waste degradation products, demonstrating that simply adding M9 salts can effectively convert them into viable raw materials for E. coli cell factories. Our findings also emphasize the significant roles of genes associated with the glycolate and glyoxylate degradation I pathway in the metabolic utilization of ethylene glycol, an aspect frequently overlooked in previous research.

Abstract Base editing technology is an ideal solution for treating pathogenic single-nucleotide variations (SNVs). No gene editing therapy has yet been approved for eye diseases, such as retinitis pigmentosa (RP). Here, we show, in the rd10 mouse model, which carries an SNV identified as an RP-causing mutation in human patients, that subretinal delivery of an optimized dual adeno-associated virus system containing the adenine base editor corrects the pathogenic SNV in the neuroretina with up to 49% efficiency. Light microscopy showed that a thick and robust outer nuclear layer (photoreceptors) was preserved in the treated area compared with the thin, degenerated outer nuclear layer without treatment. Substantial electroretinogram signals were detected in treated rd10 eyes, whereas control treated eyes showed minimal signals. The water maze experiment showed that the treatment substantially improved vision-guided behavior. Together, we construct and validate a translational therapeutic solution for the treatment of RP in humans. Our findings might accelerate the development of base-editing based gene therapies.

Abstract C-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.

Prime editing represents a great advance to the genome editing field but is currently limited by the editing efficiency. Here the authors look to improve the efficiency by recruiting target proteins and show that the transcription factor P65 could enhance the desired editing outcomes at different gene loci.

A large number of cell disease models with pathogenic SNVs are needed. Here the authors report an automated high-throughput platform to perform the genome editing process from gRNA design to the analysis of the editing results; they characterise in situ base editing outcomes.

Abstract Background Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. Results The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli 0.10/4.54 and E. coli 2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli 0.10/4.54 was quite stable, the two chromosomes of E. coli 2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. Conclusion Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.

Abstract Glucose and xylose are two major components of lignocellulose. Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass. Although many production limitations have been resolved, glucose‐induced inhibition of xylose transport remains a challenge. In this study, a cell growth‐based screening strategy was designed to identify xylose transporters uninhibited by glucose. The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival. Through adaptive evolution, omics analysis and reverse metabolic engineering, a new phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) galactitol transporter (GalABC, encoded by EcolC_1640, EcolC_1641, and EcolC_1642 genes) that is not inhibited by glucose was identified. Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene, and a point mutation in gene EcolC_1642 (N13S) further enhanced xylose transport. During the second round of gene mining, AraE and a new ABC transporter (AraFGH) of xylose were identified. A point mutation in the transcription regulator araC (L156I) caused increased expression of araE and araFGH genes without arabinose induction, and a point mutation in araE (D223Y) further enhanced xylose transport. These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

Abstract Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR‐based chromosome‐doubling technique is designed to first construct an artificial diploid Escherichia coli cell. The stable single‐cell diploid E. coli is isolated by both maximal dilution plating and flow cytometry, and confirmed with quantitative PCR, fluorescent in situ hybridization, and third‐generation genome sequencing. The diploid E. coli has a greatly reduced growth rate and elongated cells at 4–5 µm. It is robust against radiation, and the survival rate after exposure to UV increased 40‐fold relative to WT. As a novel life form, the artificial diploid E. coli is an ideal substrate for research fundamental questions in life science concerning polyploidy. And this technique may be applied to other bacteria.

Abstract Base editing events in eukaryote require a compatible chromatin environment, but there is little research on how chromatin factors contribute to the editing efficiency or window. By engineering BEs (base editors) fused with various pioneer factors, the authors found that SOX2 substantially increased the editing efficiency for GBE and CBE. While SoxN‐GBE (SOX2‐NH3‐GBE) improved the editing efficiency at overall cytosines of the protospacer, SoxM‐GBE/CBE (SOX2‐Middle‐GBE/CBE) enabled the higher base editing at PAM‐proximal cytosines. By separating functional domains of SOX2, the SadN‐GBE (SOX2 activation domain‐NH3‐GBE) is constructed for higher editing efficiency and SadM‐CBE for broader editing window to date. With the DNase I assay, it is also proved the increased editing efficiency is most likely associated with the induction of chromatin accessibility by SAD. Finally, SadM‐CBE is employed to introduce a stop codon in the proto‐oncogene MYC, at a locus rarely edited by previous editors with high efficiency. In this work, a new class of pioneer‐BEs is constructed by fusion of pioneer factor or its functional domains, which exhibits higher editing efficiency or broader editing window in eukaryote.

Summary The unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di‐ and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid‐tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2‐1 and PkJEN2‐2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L‐malate into the cell. In addition, PkJEN2‐1 can transport α‐ketoglutarate, while PkJEN2‐2 cannot. Since PkJEN2‐1 shows higher transcript abundance than PkJEN2‐2, its role in dicarboxylate transport is more important than PkJEN2‐2. In addition, PKJEN2‐2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model‐based structure analysis and rational mutagenesis further proved that amino acid residues 392‐403 of the tenth transmembrane span (TMS‐X) of PkJEN2‐2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di‐/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis.

Abstract Background CO2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin–Benson–Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO2 fixation efficiency of cells is an important subject of biological research with significant application potential. Results In this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD600 of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD600 to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%. Conclusions To our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO2 assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.

Abstract Background Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment. Results Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpF-Cas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components. Conclusion This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location.

Abstract Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21’s defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21’s to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.

Abstract Background Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones. Results In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

Abstract Background Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. Results In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC–ESI–MS. Conclusions A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Abstract Background Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO2. Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products. Results In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO3 +, which produced a succinate yield of 1.10 mol/mol glucose—a 1.6-fold improvement over the parent strain T110. Conclusions The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.

ABSTRACT Biofilms usually form when the density of bacteria increases during the middle to late periods of growth in culture, commonly induced by quorum-sensing systems. Biofilms attach to the surfaces of either living or nonliving objects and protect bacteria against antibiotics and a host’s immune system. Here, a novel type of biofilm (the “R-biofilm”) is reported. These biofilms were formed by clinically isolated Klebsiella pneumoniae strains following double-stranded-DNA breaks (DSBs), while undamaged bacteria did not form classic biofilms even in the later stages of growth. R-biofilms had a fixed ring-like or discoid shape with good ductility and could protect many living bacterial cells within. We show that extracellular proteins and DNAs released, probably by dead bacteria, were the core structural materials of R-biofilms. We anticipate that novel signaling pathways besides the bacterial SOS response are involved in R-biofilm formation. The observations in this study suggest a limitation to the use of the currently popular Cas9-mediated bactericidal tools to eliminate certain bacteria because the resulting DSBs may lead to the formation of these protective R-biofilms. IMPORTANCE Many pathogenic bacteria can form biofilm matrices that consist of complex molecules such as polysaccharides, proteins, and DNA. These biofilms help the bacteria to infect and colonize a host. Such biofilms may attach and develop on the surfaces of indwelling medical devices or other supportive environments. This study found that following double-strand breaks in their DNA, Klebsiella pneumoniae cells can form a novel type of biofilm with ring-like or discoid morphology. This biofilm structure, named the “R-biofilm,” helps protect the bacteria against adverse conditions such as exposure to ethanol, hydrogen peroxide, and UV radiation.

Abstract Background One important metabolic engineering strategy is to localize the enzymes close to their substrates for improved catalytic efficiency. However, localization configurations become more complex the greater the number of enzymes and substrates is involved. Indeed, optimizing synthetic pathways by localizing multiple enzymes remains a challenge. Terpenes are one of the most valuable and abundant natural product groups. Phytoene, lycopene and β-carotene serve as common intermediates for the synthesis of many carotenoids and derivative compounds, which are hydrophobic long-chain terpenoids, insoluble in water and usually accumulate in membrane compartments. Results While β-ionone synthesis by β-carotene cleavage dioxygenase PhCCD1 and astaxanthin synthesis by β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) differ in complexity (single and multiple step pathways), the productivity of both pathways benefited from controlling enzyme localization to the E. coli cell membrane via a GlpF protein fusion. Especially, the astaxanthin synthesis pathway comprises both CrtW and CrtZ, which perform four interchangeable reactions initiated from β-carotene. Up to four localization strategies of CrtW and CrtZ were exhaustively discussed in this work, and the optimal positioning strategy was achieved. CrtW and CrtZ were linked using a flexible linker and localized to the membrane via a GlpF protein fusion. Enzymes in the optimal localization configuration allowed a 215.4% astaxanthin production increase. Conclusions This work exploits a localization situation involving membrane-bound substrates, intermediates and multiple enzymes for the first time, and provides a workable positioning strategy to solve problems in similar circumstances.

Abstract Background Geranyl acetate is widely used in the fragrance and cosmetic industries, and thus has great economic value. However, plants naturally produce a mixture of hundreds of esters, and geranyl acetate is usually only present in trace amounts, which makes its economical extraction from plant sources practically impossible. As an ideal host for heterologous production of fragrance compound, the Saccharomyces cerevisiae has never been engineered to produce the esters, such as geranyl acetate. Results In this study, a heterologous geranyl acetate synthesis pathway was constructed in S. cerevisiae for the first time, and a titer of 0.63 mg/L geranyl acetate was achieved. By expressing an Erg20 mutant to divert carbon flux from FPP to GPP, the geranyl acetate production increased to 2.64 mg/L. However, the expression of heterologous GPP had limited effect. The highest production of 13.27 mg/L geranyl acetate was achieved by additional integration and expression of tHMG1, IDI1 and MAF1. Furthermore, through optimizing fermentation conditions, the geranyl acetate titer increased to 22.49 mg/L. Conclusions We constructed a monoterpene ester producing cell factory in S. cerevisiae for the first time, and demonstrated the great potential of this system for the heterologous production of a large group of economically important fragrance compounds.

Abstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. Results An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.

Abstract Background Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application. Results In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose. Conclusions With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Abstract A balanced and optimized metabolic pathway is the basis for efficient production of a target metabolite. Traditional strategies mostly involve the manipulation of promoters or ribosome‐binding sites, which can encompass long sequences and can be complex to operate. In this work, we found that by changing only the three nucleotides of the initiation codons, expression libraries of reporter proteins RFP, GFP, and lacZ with a large dynamic range and evenly distributed expression levels could be established in Escherichia coli (E. coli). Thus, a novel strategy that uses combinatorial modulation of initial codons (CMIC) was developed for metabolic pathway optimization and applied to the three genes crtZ, crtY, and crtI of the zeaxanthin synthesis pathway in E. coli. The initial codons of these genes were changed to random nucleotides NNN, and the gene cassettes were assembled into vectors via an optimized strategy based on type II restriction enzymes. With minimal labor time, a combinatorial library was obtained containing strains with various zeaxanthin production levels, including a strain with a titer of 6.33 mg/L and specific production value of 1.24 mg/g DCW—a striking 10‐fold improvement over the starting strain. The results demonstrated that CMIC was a feasible technique for conveniently optimizing metabolic pathways. To our best knowledge, this is the first metabolic engineering strategy that relies on manipulating the initiation codons for pathway optimization in E. coli.

The PET operon containing the pdc, adhA, and adhB genes from Zymomonas mobilis is incorporated into a pflB Enterobacter asburiae JDR-1 isolate by plasmid transformation to construct homoethanologenic strains. The resulting recombinant strains are compared with Enterobacter asburiae wild-type strain JDR-1 and the ethanologenic strain Escherichia coli KO11 to analyze their efficiencies of production of ethanol from dilute acid hydrolysates of sweet gum methylglucuronoxylans (MeGA[X.sub.n]).

The isolation and characterization of a bacterium, Enterobacter asburiae strain JDR-1, which has the ability to ferment aldobiuronate methylglucuronoxylose (MeGAX) as the sole carbon source is described. Enterobacter asburiae JDR-1 is defined with respect to its metabolic potential for converting MeGAX, xylose and glucose to specific fermentation products.

The bypass operation method of mobile transformer vehicles is a common method for the maintenance or replacement of transformers in distribution lines. Using the bypass operation method could avoid power outages and ensure the reliability of electricity consumption. However, the transient characteristics of mobile transformer vehicles connected to the grid can have an impact on their power quality. In response to this problem, this study built a model based on MATLAB/Simulink to model the process of grid connection. The simulation analyzed the current and voltage waveforms of the transient process of grid connection. Moreover, a combination of time-domain and frequency-domain analysis was used to analyze the transient overvoltage and overcurrent conditions of the grid connection. Finally, the effects of two overcurrent suppression methods, adjustment of the closing angle method and the series closing resistance method, were analyzed. These studies provide theoretical guidance for engineering applications, which could strengthen the stability of the distribution network.

Background: One of the most important research subjects of metabolic engineering is pursuing a balanced metabolic pathway, which is the basis of an efficient cell factory. In this work, we dedicated to develop a simple and efficient technique to modulate expression of multiple genes simultaneously, and select for the optimal regulation pattern. Results: A Type IIs restriction based combinatory modulation (TRCM) technique was designed and established in the research. With this technique, a plasmid library containing variably regulated mvaE, mvaS, mvaK1, mvaD and mvaK2 of the mevalonate (MVA) pathway were obtained and transformed into E. coli DXS37-IDI46 to obtain a β-carotene producer library. The ratio of successfully assembled plasmids was determined to be 35%, which was increased to 100% when color based pre-screening was applied. Representative strains were sequenced to contain diverse RBSs as designed to regulate expression of MVA pathway genes. A relatively balanced MVA pathway was achieved in E. coli cell factory to increase the β-carotene yield by two fold. Furthermore, the approximate regulation pattern of this optimal MVA pathway was illustrated. Conclusions: A TRCM technique for metabolic pathway optimization was designed and established in this research, which can be applied to various applications in terms of metabolic pathway regulation and optimization. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. Results: Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype. Conclusions: The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production. [ABSTRACT FROM AUTHOR]

Background: Plasmid expression is a popular method in studies of MVA pathway for isoprenoid production in Escherichia coli. However, heterologous gene expression with plasmid is often not stable and might burden growth of host cells, decreases cell mass and product yield. In this study, MVA pathway was divided into three modules, and two heterologous modules were integrated into the E. coli chromosome. These modules were individually modulated with regulatory parts to optimize efficiency of the pathway in terms of downstream isoprenoid production. Results: MVA pathway modules Hmg1-erg12 operon and mvaS-mvaA-mavD1 operon were integrated into E. coli chromosome followed by modulation with promoters with varied strength. Along with activation of atoB, a 26% increase of β-carotene production with no effect on cell growth was obtained. With a combinatory modulation of two key enzymes mvas and Hmg1 with degenerate RBS library, β-carotene showed a further increase of 51%. Conclusions: Our study provides a novel strategy for improving production of a target compound through integration and modulation of heterologous pathways in both transcription and translation level. In addition, a genetically hard-coded chassis with both efficient MEP and MVA pathways for isoprenoid precursor supply was constructed in this work. [ABSTRACT FROM AUTHOR]

Background: Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods. Results: In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h. Conclusion: In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci. [ABSTRACT FROM AUTHOR]

Background: Saccharomyces cerevisiae has already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways in S. cerevisiae became the research focus of Synthetic Biology and Metabolic Engineering. Thus, simple and efficient genomic integration techniques of large number of transcription units are demanded urgently. Results: An efficient DNA assembly and chromosomal integration method was created by combining homologous recombination (HR) in S. cerevisiae and Golden Gate DNA assembly method, designated as modularized two-step (M2S) technique. Two major assembly steps are performed consecutively to integrate multiple transcription units simultaneously. In Step 1, Modularized scaffold containing a head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled with Golden Gate method into one scaffold in one reaction. In Step 2, the two transcription units were mixed with modules of selective markers and integration sites and transformed into S. cerevisiae for assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway in S. cerevisiae within 5 days demonstrated high efficiency of this method, and a 10-transcriptional-unit pathway integration illustrated the capacity of this method. Conclusions: Modular design of transcription units and integration elements simplified assembly and integration procedure, and eliminated frequent designing and synthesis of DNA fragments in previous methods. Also, by assembling most parts in Step 1 in vitro, the number of DNA cassettes for homologous integration in Step 2 was significantly reduced. Thus, high assembly efficiency, high integration capacity, and low error rate were achieved. [ABSTRACT FROM AUTHOR]

Background: Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. Results: Herein, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform's capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis. Conclusions: Taken together, we demonstrate the microfluidic platform's potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis. [ABSTRACT FROM AUTHOR]

Background The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well. [ABSTRACT FROM AUTHOR]

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G in vivo mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10-3 and 1.20 × 10-2, respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great in vivo evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox. [ABSTRACT FROM AUTHOR]

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones. [ABSTRACT FROM AUTHOR]

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes ( LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B , and SPINK6 ) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC , and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.