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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

High-resolution mapping and genetic characterization of the Lazy-2 gravitropic mutant of tomato

Mutation of the Lazy-2 (Lz-2) gene in tomato (Lycopersicon esculentum mill.) produces a phytochrome-dependent reversal of shoot gravitropism, providing a unique genetic resource for investigating how signals from light modulate gravitropism. We mapped the Lz-2 gene using RFLPs and a PCR-based technique to assess the feasibility of positional cloning. Analysis of a 1338 plant backcross population between L. esculentum and L. pennellii placed Lz-2 within a 1.2 cM interval on chromosome 5, 0.4 cM from TG504-CT201A interval. The inabililty to resolve these markers indicates that Lz-2 resides in a centromeric region in which recombination is highly suppressed. Lazy-2 is tightly linked to but does not encode the gene for ACC4, an enzyme involved in ethylene biosynthesis. We also observed that Lz-2 is partially dominant under certain conditions and stages of development.

Non-NASA Center↗
DOE OSTI2020

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas

Abstract Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

Neupert, Juliane↗
DOE OSTI2021

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain ?cre-1, while glucose repression was still mostly functional in ?exo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in ?exo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in ?exo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Gabriel, Raphael↗
DOE OSTI2025

A small number of point mutations confer formate tolerance in Shewanella oneidensis

ABSTRACT Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO 2 and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology. Shewanella oneidensis could serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO 2 reduction to formate. However, we report that wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a gene encoding a predicted sodium-dependent bicarbonate transporter and a DUF2721-containing protein separately confer formate tolerance to S. oneidensis . The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of the putative sodium-dependent bicarbonate transporter improve formate tolerance of Zymomonas mobilis , indicating the potential of transferring this formate tolerance phenotype to other organisms. IMPORTANCE Shewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO 2 or formate as feedstocks. However, we report here that formate is toxic to S. oneidensis , limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that have improved formate tolerance, and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild-type and mutant versions of each gene to the wild-type strain. Finally, the formate tolerance mechanism of one variant is studied using structural modeling and expression in another host. This study, therefore, presents a simple method for conferring formate tolerance to bacterial hosts.

Cross, Megan C. Gruenberg (ORCID:0000000291589900)↗
DOE OSTI2023

Comparative genomics of the Liberibacter genus reveals widespread diversity in genomic content and positive selection history

‘Candidatus Liberibacter’ is a group of bacterial species that are obligate intracellular plant pathogens and cause Huanglongbing disease of citrus trees and Zebra Chip in potatoes. Here, we examined the extent of intra- and interspecific genetic diversity across the genus using comparative genomics. Our approach examined a wide set of Liberibacter genome sequences including five pathogenic species and one species not known to cause disease. By performing comparative genomics analyses, we sought to understand the evolutionary history of this genus and to identify genes or genome regions that may affect pathogenicity. With a set of 52 genomes, we performed comparative genomics, measured genome rearrangement, and completed statistical tests of positive selection. We explored markers of genetic diversity across the genus, such as average nucleotide identity across the whole genome. These analyses revealed the highest intraspecific diversity amongst the ‘Ca. Liberibacter solanacearum’ species, which also has the largest plant host range. We identified sets of core and accessory genes across the genus and within each species and measured the ratio of nonsynonymous to synonymous mutations (dN/dS) across genes. We identified ten genes with evidence of a history of positive selection in the Liberibacter genus, including genes in the Tad complex, which have been previously implicated as being highly divergent in the ‘Ca. L. capsica’ species based on high values of dN.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae : Inactivation of the VesC Protease

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae. Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2005

Development and Function of the Mouse Vestibular System in the Absence of Gravity Perception

The hypothesis that was tested in this research was that the absence of gravity perception, such as would occur in space, would affect the development and function of the vestibular and central nervous systems. Further, we postulated that these effects would be more significant at specific stages of post-natal development of the animal. We also proposed the use of molecular genetic approaches that would provide important information as to the hierarchy of gene function during the development and subsequent function of the vestibular system. The tilted (tlt) mutant mouse has been characterized as lacking the ability to provide sensory input to the gravity receptors. The tlt/tlt mutant mice were a particularly attractive model for the study of vestibular function since the primary defect was limited to the receptor part of the vestibular system, and there were no detectable abnormal phenotypes in other organ systems. The goal of the proposed studies was to assess immediate and delayed effects of the lack of gravity perception on the vestibular system. Particular attention was paid to characterizing primarily affected periods of vestibular morphogenesis, and to identifying downstream genetic pathways that are altered in the CNS of the tlt/tlt mutant mouse. The specific aims were: (1) to characterize the postnatal morphogenesis of the CNS in the tlt mutant mouse, using detailed morphometric analysis of isolated vestibular ganglia and brain tissue at different stages of postnatal development and assessment of apoptotic cell death; (2) to examine the expression of selected genes implicated by mutational analysis to be important in vestibular development or function by in situ hybridization or immunohistochemistry in the mutant mice; and (3) to identify other genes involved in vestibular development and function, using differential cloning strategies to isolate genes whose expression is changed in the mutant versus normal vestibular system.

Wolgemuth, Debra J.↗
DOE OSTI2021

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. In this work, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon

Background: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. Results: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. Conclusion: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The Adaptive Potential of the Middle Domain of Yeast Hsp90

Abstract The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same ≈2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase–chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase–chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information.

Cote-Hammarlof, Pamela A.↗
NASA NTRSDate not supplied

Guanine Oxidation in the Genome, not RNA Editing, Accounts for Single Nucleotide Variation in the Exome of Mice Flown on Board the ISS

We have conducted a further analysis of single nucleotide variation (somatic mutation) in mice flown aboard the ISS. We used data archived in GeneLab from a cohort of 18-week-old mice were flown to the ISS, housed in the Rodent Habitat and therefore subjected to microgravity for 37 days. Mice of similar age, sex and the same strain were used for ground controls housed in identical hardware and simulating, but not matching ISS environmental conditions (temperature, humidity and gas atmosphere). Primary data consists of next generation RNA sequencing for the tissues examined: eye, liver, skeletal muscle and kidney. We used novel software, developed at NASA Ames Research Center and deployed on the NASA Ames Supercomputer, to perform variant calling for single point mutations. Unexpectedly, we discovered a high degree of somatic mutation in ISS-flown mice, compared to controls. We found that the extent of somatic mutation correlated with the degree of gene expression in the four tissue types, with the highest degree of somatic mutation observed in genes with the highest degree of expression. Careful analysis that included measurement of specific nucleotide changes that occurred demonstrated that guanine substitutions were the most frequent, consistent with the hypothesis that reactive oxygen species-mediated guanine oxidation was responsible for the hypermutation events. By contrast, adenine substitutions would be expected if gene editing were responsible for the somatic mutation. These types of substitutions were much less frequent. The implication of these findings for astronaut health in a variety of mission scenarios will be discussed.

International Space Station↗
DOE OSTI2021

Attaining the promise of plant gene editing at scale

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Designing a Unique Single Point Cross Over Method

The idea behind genetic algorithms is to extract optimization strategies nature uses successfully - known as Darwinian Evolution - and transform them for application in mathematical optimization theory to find the global optimum in a defined phase space. One could imagine a population of individual 'explorers' sent into the optimization phase-space. Each explorer is defined by its genes, what means, its position inside the phase-space is coded in his genes. Every explorer has the duty to find a value of the quality of his position in the phase space. (Consider the phase-space being a number of variables in some technological process, the value of quality of any position in the phase space - in other words: any set of the variables - can be expressed by the yield of the desired chemical product.) Then the struggle of 'life' begins. The three fundamental principles are selection, mating/crossover, and mutation. Only explorers (= genes) sitting on the best places will reproduce and create a new population. This is performed in the second step (mating/crossover). The 'hope' behind this part of the algorithm is, that 'good' sections of two parents will be recombined to yet better fitting children. In fact, many of the created children will not be successful (as in biological evolution), but a few children will indeed fulfill this hope. These good sections are named in some publications as building blocks. Now there appears a problem. Repeating these steps, no new area would be explored. The two former steps would only exploit the already known regions in the phase space, which could lead to premature convergence of the algorithm with the consequence of missing the global optimum by exploiting some local optimum. The third step, mutation, ensures the necessary accidental effects. One can imagine the new population being mixed up a little bit to bring some new information into this set of genes. Whereas in biology a gene is described as a macro-molecule with four different bases to code the genetic information, a gene in genetic algorithms is usually defined as a bitstring (a sequence of b 1's and 0's).

Wilson, Richard Phillip↗
DOE OSTI2023

Two disulfide-reducing pathways are required for the maturation of plastid c -type cytochromes in Chlamydomonas reinhardtii

Abstract In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f.

Genetics & Heredity↗
DOE OSTI2024

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

Abstract Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt‐loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme‐coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum , TrmB‐dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii . The ∆ trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name t rm B s u p pressor, or TbsP (a.k.a. “tablespoon”). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild‐type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose‐dependent transcription of gapII , which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii .

Biochemistry & Molecular Biology↗
DOE OSTI2020

Effects of Genetic and Physiological Divergence on the Evolution of a Sulfate-Reducing Bacterium under Conditions of Elevated Temperature

ABSTRACT Adaptation via natural selection is an important driver of evolution, and repeatable adaptations of replicate populations, under conditions of a constant environment, have been extensively reported. However, isolated groups of populations in nature tend to harbor both genetic and physiological divergence due to multiple selective pressures that they have encountered. How this divergence affects adaptation of these populations to a new common environment remains unclear. To determine the impact of prior genetic and physiological divergence in shaping adaptive evolution to accommodate a new common environment, an experimental evolution study with the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was conducted. Two groups of replicate populations with genetic and physiological divergence, derived from a previous evolution study, were propagated in an elevated-temperature environment for 1,000 generations. Ancestor populations without prior experimental evolution were also propagated in the same environment as a control. After 1,000 generations, all the populations had increased growth rates and all but one had greater fitness in the new environment than the ancestor population. Moreover, improvements in growth rate were moderately affected by the divergence in the starting populations, while changes in fitness were not significantly affected. The mutations acquired at the gene level in each group of populations were quite different, indicating that the observed phenotypic changes were achieved by evolutionary responses that differed between the groups. Overall, our work demonstrated that the initial differences in fitness between the starting populations were eliminated by adaptation and that phenotypic convergence was achieved by acquisition of mutations in different genes. IMPORTANCE Improving our understanding of how previous adaptation influences evolution has been a long-standing goal in evolutionary biology. Natural selection tends to drive populations to find similar adaptive solutions for the same selective conditions. However, variations in historical environments can lead to both physiological and genetic divergence that can make evolution unpredictable. Here, we assessed the influence of divergence on the evolution of a model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, in response to elevated temperature and found a significant effect at the genetic but not the phenotypic level. Understanding how these influences drive evolution will allow us to better predict how bacteria will adapt to various ecological constraints.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis

Previously, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. In this paper, we developed a protoplast assay to rapidly optimize the multiplexed CRISPR/Cas9 system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples with 94% of deletions larger than 10bp, and less than 1% of mutations being insertions. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical NHEJ DNA repair pathway. Combined with the robust agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at the frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable MMEJ-mediated mutations in many plant species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803

Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.

59 BASIC BIOLOGICAL SCIENCES↗