FIT2 proteins and lipid droplet emergence, an interplay between phospholipid synthesis, surface tension, and membrane curvature.

Lipid droplets (LDs) serve as intracellular compartments primarily dedicated to the storage of metabolic energy in the form of neutral lipids. The processes that regulate and control LD biogenesis are being studied extensively and are gaining significance due to their implications in major metabolic disorders, including type 2 diabetes and obesity. A protein of particular interest is Fat storage-Inducing Transmembrane 2 (FIT2), which affects the emergence step of LD biogenesis. Instead of properly emerging towards the cytosol, LDs in FIT2-deficient cells remain embedded within the membrane of the endoplasmic reticulum (ER). In vitro studies revealed the ability of FIT2 to bind both di- and triacylglycerol (DAG/TAG), key players in lipid storage, and its activity to cleave acyl-CoA. However, the translation of these in vitro functions to the observed embedding of LDs in FIT2 deficient cells remains to be established. To understand the role of FIT2 in vivo, we discuss the parameters that affect LD emergence. Our focus centers on the role that membrane curvature and surface tension play in LD emergence, as well as the impact that the lipid composition exerts on these key parameters. In addition, we discuss hypotheses on how FIT2 could function locally to modulate lipids at sites of LD emergence.

Metabolites of pathogenic microorganisms database (MPMdb) and its seed metabolite applications.

Seed metabolites are the combination of essential compounds required by an organism across various potential environmental conditions. The seed metabolites screening framework based on the network topology approach can capture important biological information of species. This study aims to identify comprehensively the relationship between seed metabolites and pathogenic bacteria. A large-scale data set was compiled, describing the seed metabolite sets and metabolite sets of 124,192 pathogenic strains from 34 genera, by constructing genome-scale metabolic models. The enrichment analysis method was used to screen the specific seed metabolites of each species/genus of pathogenic bacteria. The metabolites of pathogenic microorganisms database (MPMdb) (http://qyzhanglab.hzau.edu.cn/MPMdb/) was established for browsing, searching, predicting, or downloading metabolites and seed metabolites of pathogenic microorganisms. Based on the MPMdb, taxonomic and phylogenetic analyses of pathogenic bacteria were performed according to the function of seed metabolites and metabolites. The results showed that the seed metabolites could be used as a feature for microorganism chemotaxonomy, and they could mirror the phylogeny of pathogenic bacteria. In addition, our screened specific seed metabolites of pathogenic bacteria can be used not only for further tapping the nutritional resources and identifying auxotrophies of pathogenic bacteria but also for designing targeted bactericidal compounds by combining with existing antimicrobial agents.IMPORTANCEMetabolites serve as key communication links between pathogenic microorganisms and hosts, with seed metabolites being crucial for microbial growth, reproduction, external communication, and host infection. However, the large-scale screening of metabolites and the identification of seed metabolites have always been the main technical bottleneck due to the low throughput and costly analysis. Genome-scale metabolic models have become a recognized research paradigm to investigate the metabolic characteristics of species. The developed metabolites of pathogenic microorganisms database in this study is committed to systematically predicting and identifying the metabolites and seed metabolites of pathogenic microorganisms, which could provide a powerful resource platform for pathogenic bacteria research.

Characterizing a panel of amino acid auxotrophs under auxotrophic starvation conditions.

Auxotrophic strains starving for their cognate nutrient, termed auxotrophic starvation, are characterized by a shorter lifespan, higher glucose wasting phenotype, and inability to accomplish cell cycle arrest when compared to a "natural starvation," where a cell is starving for natural environmental growth-limiting nutrients such as phosphate. Since evidence of this physiological response is limited to only a subset of auxotrophs, we evaluated a panel of auxotrophic mutants to determine whether these responses are characteristic of a broader range of amino acid auxotrophs. Based on the starvation survival kinetics, the panel of strains was grouped into three categories-short-lived strains, strains with survival similar to a prototrophic wild type strain, and long-lived strains. Among the short-lived strains, we observed that the tyrosine, asparagine, threonine, and aspartic acid auxotrophs rapidly decline in viability, with all strains unable to arrest cell cycle progression. The three basic amino acid auxotrophs had a survival similar to a prototrophic strain starving in minimal media. The leucine, tryptophan, methionine, and cysteine auxotrophs displayed the longest lifespan. We also demonstrate how the phenomenon of glucose wasting is limited to only a subset of the tested auxotrophs, namely the asparagine, leucine, and lysine auxotrophs. Furthermore, we observed pleiotropic phenotypes associated with a subgroup of auxotrophs, highlighting the importance of considering unintended phenotypic effects when using auxotrophic strains especially in chronological aging experiments.

Complete genome of Polaribacter huanghezhanensis KCTC 32516T isolated from glaciomarine fjord sediment of Svalbard.

Polaribacter huanghezhanensis KCTC 32516T is an aerobic, non-flagellated, Gram-negative, orange-colony-forming bacterium that was isolated from the surficial glaciomarine sediment of inner basin of Kongsfjorden, Svalbard. The sampling site is characterized by a sedimentation of organic depleted lithogenous particles from the nearby glaciers, resulting in reduction of organic matter concentration. In order to understand microbial adaptation to the oligotrophic environment, we here sequenced the complete genome of the P. huanghezhanensis KCTC 32516T. The genome consists of 2,587,874 bp (G + C content of 31.5%) with a single chromosome, 2391 protein-coding genes, 39 tRNAs, and 2 rRNA operons. Our comparative analysis revealed that the P. huanghezhanensis possess the smallest genome in fifteen Polaribacter species with genome. The streamlined genome of this species, required less resource in replication, could evolved by the nutrient deficiency in surrounding environment. Simultaneously, the 15 KOs involved in amino acid biosynthesis and anaplerotic carbon fixation is uniquely absent in the P. huanghezhanensis. In addition, although the advantage of small genome, other 15 KOs involved in resource recycling and stress resistance is uniquely present in sequenced genome. This result demonstrates that the sequenced genome serves as a valuable model for further studies aimed at elucidating the molecular mechanisms associated with adaptation to oligotrophic habitat.

Tumor-targeted delivery of lnc antisense RNA against RCAS1 by live-attenuated tryptophan-auxotrophic Salmonella inhibited 4T1 breast tumors and metastasis in mice.

Emerging chemo- and radiotherapy resistance exacerbated the cancer risk and necessitated novel treatment strategies. Although RNA therapeutics against pro-oncogenic genes are highly effective, tumor-specific delivery remains a barrier to the implementation of this valuable tool. In this study, we report a tryptophan-auxotrophic Salmonella typhimurium strain as an onco-therapeutic delivery system with tumor-targeting ability using 4T1 mice breast-cancer model. The receptor-binding cancer antigen expressed on SiSo cell (RCAS1) is a cancer-specific protein that induces the apoptosis of peripheral lymphocytes and confers tumor immune evasion. We designed a long non-coding antisense-RNA against RCAS1 (asRCAS1) and delivered by Salmonella using a non-antibiotic, auxotrophic-selective, eukaryotic expression plasmid, pJHL204. After in vivo tumor-to-tumor passaging, the JOL2888 (ΔtrpA, ΔtrpE, Δasd + asRCAS1) strain exhibited high sustainability in tumors, but did not last in healthy organs, thereby demonstrating tumor specificity and safety. RCAS1 inhibition in the tumor was confirmed by western blotting and qPCR. In mice, JOL2888 treatment reduced tumor-associated macrophages, improved the T cell population, elicited cell-mediated immunity, and suppressed cancer-promoting genes. Consequently, the JOL2888 treatment significantly decreased the tumor volume by 80%, decreased splenomegaly by 30%, and completely arrested lung metastasis. These findings highlight the intrinsic tumor-targeting ability of tryptophan-auxotrophic Salmonella for delivering onco-therapeutic macromolecules.

AI-2 quorum sensing controlled delivery of cytolysin-A by tryptophan auxotrophic low-endotoxic Salmonella and its anticancer effects in CT26 mice with colon cancer.

INTRODUCTION: The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. OBJECTIVES: This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. METHODS: The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. RESULTS: The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1ß, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. CONCLUSION: Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

Growth-coupled high throughput selection for directed enzyme evolution.

Directed enzyme evolution has revolutionized the rapid development of enzymes with desired properties. However, the lack of a high-throughput method to identify the most suitable variants from a large pool of genetic diversity poses a major bottleneck. To overcome this challenge, growth-coupled in vivo high-throughput selection approaches (GCHTS) have emerged as a novel selection system for enzyme evolution. GCHTS links the survival of the host cell with the properties of the target protein, resulting in a screening system that is easily measurable and has a high throughput-scale limited only by transformation efficiency. This allows for the rapid identification of desired variants from a pool of >109 variants in each experiment. In recent years, GCHTS approaches have been extensively utilized in the directed evolution of multiple enzymes, demonstrating success in catalyzing non-native substrates, enhancing catalytic activity, and acquiring novel functions. This review introduces three main strategies employed to achieve GCHTS: the elimination of toxic compounds via desired variants, enabling host cells to thrive in hazardous conditions; the complementation of an auxotroph with desired variants, where essential genes for cell growth have been eliminated; and the control of the transcription or expression of a reporter gene related to host cell growth, regulated by the desired variants. Additionally, we highlighted the recent developments in the in vivo continuous evolution of enzyme technology, including phage-assisted continuous evolution (PACE) and orthogonal DNA Replication (OrthoRep). Furthermore, this review discusses the challenges and future prospects in the field of growth-coupled selection for protein engineering.

A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris.

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in P. pastoris. Firstly, we improved the HR rates of P. pastoris through knocking out the non-homologous-end-joining gene (Δku70) and overexpressing HR-related proteins (RAD52 and RAD59), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes PpURA3 as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter (scFUR4) to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in P. pastoris. The systems will contribute to P. pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.

Physiological and genomic analyses of cobalamin (vitamin B12)-auxotrophy of Lysobacter auxotrophicus sp. nov., a methionine-auxotrophic chitinolytic bacterium isolated from chitin-treated soil.

A novel bacterium, designated 5-21aT, isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B12)(Cbl)-auxotrophic property of strain 5-21aT. The newly determined complete genomic sequence indicated that strain 5-21aT possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21aT. The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21aT, which explains the Cbl-auxotrophy of 5-21aT. This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21aT indicated the highest similarities to Lysobacter soli DCY21T(99.8 and 99.9 %) and Lysobacter panacisoli CJ29T(98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C15:0, iso-C16:0 and iso-C17:1 ω9c. The complete genome sequence of strain 5-21aT revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21aT and its most closely phylogenetic relative L. soli DCY21T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21aT represents a novel species in the genus Lysobacter, for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21aT (=NBRC 115507T=LMG 32660T).

Mitochondrial Metabolomics of Sym1-Depleted Yeast Cells Revealed Them to Be Lysine Auxotroph.

Metabolomics has expanded from cellular to subcellular level to elucidate subcellular compartmentalization. By applying isolated mitochondria to metabolome analysis, the hallmark of mitochondrial metabolites has been unraveled, showing compartment-specific distribution and regulation of metabolites. This method was employed in this work to study a mitochondrial inner membrane protein Sym1, whose human ortholog MPV17 is related to mitochondria DNA depletion syndrome. Gas chromatography-mass spectrometry-based metabolic profiling was combined with targeted liquid chromatography-mass spectrometry analysis to cover more metabolites. Furthermore, we applied a workflow employing ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry with a powerful chemometrics platform, focusing on only significantly changed metabolites. This workflow highly reduced the complexity of acquired data without losing metabolites of interest. Consequently, forty-one novel metabolites were identified in addition to the combined method, of which two metabolites, 4-guanidinobutanal and 4-guanidinobutanoate, were identified for the first time in Saccharomyces cerevisiae. With compartment-specific metabolomics, we identified sym1Δ cells as lysine auxotroph. The highly reduced carbamoyl-aspartate and orotic acid indicate a potential role of the mitochondrial inner membrane protein Sym1 in pyrimidine metabolism.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Disruption of the metC Gene Affects Methionine Biosynthesis in Pectobacterium carotovorum subsp. carotovorum Pcc21 and Reduces Soft-Rot Disease.

Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine ß-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.

An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain / Una cepa recombinante NADPH-auxotrófica de Corynebacterium glutamicum y la utilizó para construir una cepa de alto rendimiento de L-leucina

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.(AU)

An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain.

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

Identification of genetic elements required for Listeria monocytogenes growth under limited nutrient conditions and virulence by a screening of transposon insertion library.

Listeria monocytogenes, the causative agent of listeriosis, displays a lifestyle ranging from saprophytes in the soil to pathogenic as a facultative intracellular parasite in host cells. In the current study, a random transposon (Tn) insertion library was constructed in L. monocytogenes strain F2365 and screened to identify genes and pathways affecting in vitro growth and fitness in minimal medium (MM) containing different single carbohydrate as the sole carbon source. About 2,000 Tn-mutants were screened for impaired growth in MM with one of the following carbon sources: glucose, fructose, mannose, mannitol, sucrose, glycerol, and glucose 6-phosphate (G6P). Impaired or abolished growth of L. monocytogenes was observed for twenty-one Tn-mutants with disruptions in genes encoding purine biosynthesis enzymes (purL, purC, purA, and purM), pyrimidine biosynthesis proteins (pyrE and pyrC), ATP synthase (atpI and atpD2), branched-chain fatty acids (BCFA) synthesis enzyme (bkdA1), a putative lipoprotein (LMOF2365_2387 described as LP2387), dUTPase family protein (dUTPase), and two hypothetical proteins. All Tn-mutants, except the atpD2 mutant, grew as efficiently as wild-type strain in a nutrient rich media. The virulence of twenty-one Tn-mutants was assessed in mice at 72 h following intravenous (IV) infection. The most attenuated mutants had Tn insertions in purA, hypothetical protein (LMOf2365_0064 described as HP64), bkdA1, dUTPase, LP2387, and atpD2, confirming the important role of these genes in pathogenesis. Six Tn-mutants were then tested for ability to replicate intracellularly in murine macrophage J774.1 cells. Significant intracellular growth defects were observed in two Tn-mutants with insertions in purA and HP64 genes, suggesting that an intact purine biosynthesis pathway is important for intracellular growth of L. monocytogens. These findings may not be fully generalized to all of L. monocytogenes strains due to their genetic diversity. In conclusion, Tn-mutagenesis identified that biosynthesis of purines, pyrimidines, ATP, and BCFA are important for L. monocytogens pathogenesis. Purine and pyrimidine auxotrophs play an important role in the pathogenicity in other bacterial pathogens, but our study also revealed new proteins essential for both growth in MM and L. monocytogenes strain F2365 virulence.

Live, Genetically Attenuated, Cold-Chain-Free Cholera Vaccine-A Research and Development Journey: Light at the End of a Long Tunnel.

Cholera, a diarrheal disease caused by Vibrio cholerae (V. cholerae) O139 and O1 strains, remains a public health problem. The existing World Health Organization (WHO)-licenced, killed, multiple-dose oral cholera vaccines demand 'cold-chain supply' at 2 °C-8 °C. Therefore, a live, single-dose, cold-chain-free vaccine would relieve significant bottlenecks and costs of cholera vaccination campaigns. Our cholera vaccine development journey started in 2000 at Universiti Sains Malaysia with isolation of the hemA gene from V. cholerae, followed by development of a gene mutant vaccine candidate VCUSM2 against V. cholerae O139 in 2006. In 2010, VCUSM2 reactogenicity was reduced by replacing its two wild-type ctxA gene copies with mutated ctxA to produce strain VCUSM14. Introducing the hemA gene into VCUSM14 created VCUSM14P, a strain with the 5-aminolaevulinic acid (ALA) prototrophic trait and excellent colonisation and immunological properties (100% protection to wild-type challenged rabbits). It was further refined in Asian Institute of Medicine, Science and Technology (AIMST University), with completion of single- and repeated-dose toxicity evaluations in 2019 in Sprague Dawley (SD) rats, followed by development of a novel cold-chain-free VCUSM14P formulation in 2020. VCUSM14P is unique for its intact cholera toxin B, a known mucosal adjuvant. The built-in adjuvant makes VCUSM14P an ideal vaccine delivery platform for emerging diseases (e.g. severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] and tuberculosis). Our vaccine formulation mimics natural infection, remains non-reactogenic and immunogenic in vivo, and protects against infection and disease. It will also cost less and be less cumbersome to distribute due to its stability at room temperature. These features could revolutionise the outreach of this and other vaccines to meet global immunisation programmes, particularly in low-resourced areas. The next stage of our journey will be meeting the requisite regulatory requirements to produce the vaccine for rollout to countries where it is most needed.

CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.

Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.

A multiauxotrophic CHO cell line for the rapid isolation of producers of diverse or high levels of recombinant proteins.

Chinese Hamster Ovary (CHO) cells are widely used for the high-level production of recombinant proteins. We created a multiauxotrophic mutant of CHO-K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA-based genes for the eight missing activities. CHO8A cells permit: (1) selection of transfectant clones that have incorporated genes for eight or more different polypeptides, suitable for engineering complex proteins, or pathways; and (2) the single-step selection of high producers of a particular protein. The latter is achieved by simultaneous use of eight vectors, each bearing one of the eight rescue genes and a cargo protein gene. Screening as few as 10 surviving colonies yielded high producers secreting mAbs at 84 picograms per cell per day or more. CHO8A was isolated by CRISPR-Cas9 knockout of 10 genes in the pathways to pyrimidines (Dhodh, Umps, Ctps1, Ctps2, and Tyms) and purines (Paics, Atic, Impdh1, Impdh2, and Gmps).

Engineering synthetic auxotrophs for growth-coupled directed protein evolution.

Selecting desired variants from protein libraries is always a challenge for directed evolution. Engineering synthetic auxotrophs to establish a link between cell growth and protein property allows growth-coupled in vivo selection, which is high throughput and compatible with continuous evolution. In silico simulation-guided metabolic reprogramming will help in the design of customized synthetic auxotrophs.