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.

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).

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.

Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905.

Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.

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).

Septic shock caused by a carbon dioxide-dependent and extended spectrum ß-lactamase-producing Proteus mirabilis small colony variant in a long-term bedridden patient.

Here, we report a 60-year-old chronically bedridden man with cerebral palsy who had septic shock following a history of urinary tract infection with extended spectrum ß-lactamase-producing and auxotrophic Proteus mirabilis detected on blood and urine cultures. This auxotroph formed small colonies only on the blood agar at 24 h in 5% CO2, but not in the conditions without CO2, and lacked motility and some biochemical activities. The five-year history of stones in the right renal pelvis suggests chronic urinary tract infection with P. mirabilis requiring a 28-day antibiotic treatment. This paper highlights that the CO2-dependent P. mirabilis small colony variant may cause sepsis, probably due to chronic infection in uroliths, which should warrant immediate identification.

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.

Co-culturing experiments reveal the uptake of myo-inositol phosphate synthase (EC 5.5.1.4) in an inositol auxotroph of Saccharomyces cerevisiae.

BACKGROUND: Myo-Inositol Phosphate Synthase (MIP) catalyzes the conversion of glucose 6- phosphate into inositol phosphate, an essential nutrient and cell signaling molecule. Data obtained, first in bovine brain and later in plants, established MIP expression in organelles and in extracellular environments. A physiological role for secreted MIP has remained elusive since its first detection in intercellular space. To provide further insight into the role of MIP in intercellular milieus, we tested the hypothesis that MIP may function as a growth factor, synthesizing inositol phosphate in intercellular locations requiring, but lacking ability to produce or transport adequate quantities of the cell-cell communicator. This idea was experimentally challenged, utilizing a Saccharomyces cerevisiae inositol auxotroph with no MIP enzyme, permeable membranes with a 0.4 µm pore size, and cellular supernatants as external sources of inositol isolated from S. cerevisiae cells containing either wild-type enzyme (Wt-MIP), no MIP enzyme, auxotroph (Aux), or a green fluorescent protein (GFP) tagged reporter enzyme (MIP- GFP) in co- culturing experiments. RESULTS: Resulting cell densities and microscopic studies with corroborating biochemical and molecular analyses, documented sustained growth of Aux cells in cellular supernatant, concomitant with the uptakeof MIP, detected as MIP-GFP reporter enzyme. These findings revealed previously unknown functions, suggesting that the enzyme can: (1) move into and out of intercellular space, (2) traverse cell walls, and (3) act as a growth factor to promote cellular proliferation of an inositol requiring cell. CONCLUSIONS: Co-culturing experiments, designed to test a probable function for MIP secreted in extracellular vesicles, uncovered previously unknown functions for the enzyme and advanced current knowledge concerning spatial control of inositol phosphate biosynthesis. Most importantly, resulting data identified an extracellular vesicle (a non-viral vector) that is capable of synthesizing and transporting inositol phosphate, a biological activity that can be used to enhance specificity of current inositol phosphate therapeutics.

Insect Sterol Nutrition: Physiological Mechanisms, Ecology, and Applications.

Insects, like all eukaryotes, require sterols for structural and metabolic purposes. However, insects, like all arthropods, cannot make sterols. Cholesterol is the dominant tissue sterol for most insects; insect herbivores produce cholesterol by metabolizing phytosterols, but not always with high efficiency. Many insects grow on a mixed-sterol diet, but this ability varies depending on the types and ratio of dietary sterols. Dietary sterol uptake, transport, and metabolism are regulated by several proteins and processes that are relatively conserved across eukaryotes. Sterol requirements also impact insect ecology and behavior. There is potential to exploit insect sterol requirements to (a) control insect pests in agricultural systems and (b) better understand sterol biology, including in humans. We suggest that future studies focus on the genetic mechanism of sterol metabolism and reverse transportation, characterizing sterol distribution and function at the cellular level, the role of bacterial symbionts in sterol metabolism, and interrupting sterol trafficking for pest control.

Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase.

N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M-1 s-1; the best variant displayed an efficiency of >106 M-1 s-1 at its optimal pH.

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

A protein constructed de novo enables cell growth by altering gene regulation.

Recent advances in protein design rely on rational and computational approaches to create novel sequences that fold and function. In contrast, natural systems selected functional proteins without any design a priori. In an attempt to mimic nature, we used large libraries of novel sequences and selected for functional proteins that rescue Escherichia coli cells in which a conditionally essential gene has been deleted. In this way, the de novo protein SynSerB3 was selected as a rescuer of cells in which serB, which encodes phosphoserine phosphatase, an enzyme essential for serine biosynthesis, was deleted. However, SynSerB3 does not rescue the deleted activity by catalyzing hydrolysis of phosphoserine. Instead, SynSerB3 up-regulates hisB, a gene encoding histidinol phosphate phosphatase. This endogenous E. coli phosphatase has promiscuous activity that, when overexpressed, compensates for the deletion of phosphoserine phosphatase. Thus, the de novo protein SynSerB3 rescues the deletion of serB by altering the natural regulation of the His operon.

Development of a pyrE-based selective system for Thermotoga sp. strain RQ7.

To fully unlock the biotechnological potentials of Thermotoga species, this study aimed to expand the genetic toolbox of Thermotoga by developing a new selective system. The developed system was composed of two components: a recipient strain bearing a deletion in its orotate phosphoribosyltransferase gene pyrE and a shuttle vector expressing a heterologous pyrE as the selectable marker. A spontaneous uracil auxotroph, T. sp. strain RQ7-15, was isolated at 70 °C with 2 mg/ml 5-fluoroorotic acid. The mutant carried a 112 bp deletion in pyrE and was a suitable recipient strain. To avoid homologous recombination, the pyrE gene from another thermophilic bacterium Caldicellulosiruptor saccharolyticus was used as the selectable marker. The gene was cloned into two Thermotoga-E. coli shuttle vectors, controlled by different promoters: the promoter of Thermus S-layer protein (P slpA ) in pDH25 and the promoter of the pyrimidine synthesis operon of T. sp. strain RQ7 (P RQ7.pyr ) in pDH28. After being introduced into the mutant strain RQ7-15 through natural transformation, both vectors allowed the host to thrive in a minimal medium. Single colonies of transformants were isolated and confirmed by polymerase chain reactions and restriction digestions. In summary, a pyrE-based selective system has been established in T. sp. strain RQ7.

A promising approach to enhance microalgae productivity by exogenous supply of vitamins.

In order to reduce the consumption of traditional fossil fuels and their impact on the environment, strategies to mitigate greenhouse gas emissions especially carbon dioxide needs exploration. Microalgae-based biofuels can be the best-fit plant based feed-stocks for diminishing a majority of the Universe's energy problems. Interestingly, the eukaryotic microalgae aid in fixation of almost 50% of the global carbon in the environment. Thus, determination of parameters that will enhance microalgal growth and productivity is crucial, if they are to be used as future renewable energy sources. A large percentage of phytoplankton species are auxotroph for one or more vitamins. These species, in turn, are also dependent upon the vitamin biosynthetic pathways for processing of these vitamins. The present study serves as a base to discuss the prevalence of vitamin auxotrophy in microalgae and the methods of its acquirement from external sources such as heterotrophic bacteria. The next section of the paper sheds light on possible species-specific symbiotic interactions among microalgae and bacteria. Lastly is the discussion on how heterotrophic bacteria can act as a vitamin prototroph for an explicit microalgal vitamin auxotroph. The overall focus is placed upon harnessing these symbiotic interactions with intentions to obtain enhancements in microalgal biomass, lipid productivity, and flocculation rates. Moreover, the growth and distribution of a microalgal cell that thrives on a specific vitamin is perhaps met by growing it with the bacterial communities that nourish it. Thus, possibly by ecologically engineering a potential species-specific microalgal-bacterial consortium, it could tremendously contribute to the acceleration of photosynthetic activity, microalgal productivity, exchange of primary metabolites and other biogeochemical nutrients within the mini ecosystem.

Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis.

Bioassay for amino acid quantification is an important technology for a variety of fields, which allows for easy, inexpensive, and high-throughput analyses. Here, we describe a novel translation-dependent bioassay for the quantification of amino acids. For this, the gene encoding firefly luciferase was introduced into Lactococcus lactis auxotrophic to Glu, His, Ile, Leu, Pro, Val, and Arg. After a preculture where luciferase expression was repressed, the cells were mixed with analytes, synthetic medium, and an inducer for luciferase expression. Luminescence response to the target amino acid appeared just after mixing, and linear standard curves for these amino acids were obtained during 15-60-min incubation periods. The rapid quantification of amino acids has neither been reported in previous works on bioassays nor is it theoretically feasible with conventional methods, which require incubation times of more than 4 h to allow for the growth of the microbe used. In contrast, our assay was shown to depend on protein translation, rather than on cell growth. Furthermore, replacement of the luciferase gene with that of the green fluorescent protein (GFP) or ß-galactosidase allowed for fluorescent and colorimetric detection of the amino acids, respectively. Significantly, when a Gln-auxotrophic Escherichia coli mutant was created and transformed by a luciferase expression plasmid, a linear standard curve for Gln was observed in 15 min. These results demonstrate that this methodology can provide versatile bioassays by adopting various combinations of marker genes and host strains according to the analytes and experimental circumstances.

Genetic drift opposes mutualism during spatial population expansion.

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, because genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast Saccharomyces cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.

Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding.

The uracil auxotrophic monokaryotic strain 423-9 of Lentinula edodes was crossed with nine monokaryons (cro2-2-9, W66-1, xd2-3-2, QingKe 20A, 241-1-1, 9015-1, L66-2, 241-1-2, and Qing 23A) derived from wild type strains of L. edodes. Nine dikaryotic hybrids were established from these crosses. These hybrids were fruited and 496 single spore isolates were obtained. Among these single spore isolates, 166 were identified as monokaryons under a microscope. We screened these monokaryons on selective medium and obtained 19 uracil auxotrophic monokaryons. By using the Monkaryon-monkaryon crossing method among the uracil auxotrophic monokaryons, 56 uracil auxotrophic dikaryotic strains were established on selective medium. These dikaryotic strains were unable to grow on minimal medium without uracil and exhibited slow growth rates on PDA plates compared to the wild type strain. The uracil auxotrophic dikaryotic strains also showed more vigorous growth on sawdust cultivation medium containing uracil than that without uracil. The fruiting tests showed that they formed normal fruiting bodies on the sawdust medium containing uracil. The results show that the uracil auxotrophic dikaryotic strain of L. edodes could be produced by mating, and will provide a valuable resource for future genetic studies and for spawn protection and identification.

Limitation of thiamine pyrophosphate supply to growing Escherichia coli switches metabolism to efficient D-lactate formation.

Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070. This was achieved by inactivation of thiE, a gene encoding a thiamine phosphate synthase for biosynthesis of thiamine monophosphate. The resultant mutant B0013-080A failed to grow on the medium in the absence of thiamine yet growth was restored when exogenous thiamine was provided. A linear relationship between cell mass formation and amount of thiamine supplemented was mathematically determined in a shake flask experiment and confirmed in a 7-L bioreactor system. This calculation revealed that ∼ 95-96 thiamine molecules per cell were required to satisfy cell growth. This relationship was employed to develop a novel fermentation process for D-lactate production by using thiamine as a limiting condition. A D-lactate productivity of 4.11 g · L(-1) · h(-1) from glycerol under microaerobic condition and 3.66 g · L(-1) · h(-1) from glucose under anaerobic condition was achieved which is 19.1% and 10.2% higher respectively than the parental strain. These results revealed a convenient and reliable method to control cell growth and improve D-lactate fermentation. This control strategy could be applied to other biotechnological processes that require optimal allocation of carbon between cell growth and product formation.

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.