Potential Metabolic Engineering Targets Research Articles

ECMpy 2.0: A Python package for automated construction and analysis of enzyme-constrained models

Genome-scale metabolic models (GEMs) have been widely employed to predict microorganism behaviors. However, GEMs only consider stoichiometric constraints, leading to a linear increase in simulated growth and product yields as substrate uptake rates rise. This divergence from experimental measurements prompted the creation of enzyme-constrained models (ecModels) for various species, successfully enhancing chemical production. Building upon studies that allocate macromolecule resources, we developed a Python-based workflow (ECMpy) that constructs an enzyme-constrained model. This involves directly imposing an enzyme amount constraint in GEM and accounting for protein subunit composition in reactions. However, this procedure demands manual collection of enzyme kinetic parameter information and subunit composition details, making it rather user-unfriendly. In this work, we've enhanced the ECMpy toolbox to version 2.0, broadening its scope to automatically generate ecGEMs for a wider array of organisms. ECMpy 2.0 automates the retrieval of enzyme kinetic parameters and employs machine learning for predicting these parameters, which significantly enhances parameter coverage. Additionally, ECMpy 2.0 introduces common analytical and visualization features for ecModels, rendering computational results more user accessible. Furthermore, ECMpy 2.0 seamlessly integrates three published algorithms that exploit ecModels to uncover potential targets for metabolic engineering. ECMpy 2.0 is available at https://github.com/tibbdc/ECMpy or as a pip package (https://pypi.org/project/ECMpy/).

Enhanced starch accumulation in Chlorella sorokiniana as sugar platform and the expression profiling of key regulatory proteins

Microalgae can accumulate considerable starch as a renewable sugar platform for biofuel production. In this study, Chlorella sorokiniana exhibited a significant increase in starch content from 3.0% to 49.5% DM under sulfur limitation (SL) conditions, and the lower the concentration of sulfur was, the more effective it was. Direct enzymatic hydrolysis of wet biomass resulted in a glucose yield of 97.1%, indicating its potential for fermentable sugar production. However, C. sorokiniana grew slowly, and cell division and biomass accumulation decreased under SL conditions, which could be explained by the decreased photosynthetic efficiency and protein content. The sulfur replenishment (SR) confirmed the rapid response of C. sorokiniana in starch accumulation to sulfur availability. Thus, we further investigated the expression profiling of key regulatory proteins under Control-SL-SR stages. The expression of AGPase A was strongly induced in the SL stage and subsequently downregulated during SR stage, suggesting its role as a key regulatory protein for enhanced starch accumulation. Additionally, downregulation of SBE and GBE expression likely contributed to structural modifications of the starch molecules. These proteins could serve as potential targets for metabolic engineering in further improving the starch production capacity in C. sorokiniana.

Time-resolved transcriptomic and proteomic profiling of Heyndrickxia coagulans during NaOH-buffered L-lactic acid production

The L-lactic acid (L-LA) fermentation process, based on sodium hydroxide neutralization, demonstrates environmental friendliness during product extraction. However, lactate fermentation is hindered by the pronounced stress effect of sodium lactate on the strain compared with calcium lactate. In this study, we performed time-resolved transcriptomic and proteomic analyses of Heyndrickxia coagulans DSM1 during NaOH-buffered L-LA production. The expression levels of the glycolytic genes demonstrated an initial increase followed by a subsequent decrease, whereas the tricarboxylic acid cycle genes exhibited an initial decrease followed by a subsequent increase throughout the fermentation process. Moreover, we identified clusters of genes consisting of transcription factors and ATP-binding cassette (ABC) transporters that demonstrate a progressive elevation of expression levels throughout the fermentation process, with significant upregulation observed at later stages. This investigation yields valuable insights into the response mechanisms of H. coagulans during NaOH-buffered L-LA fermentation and presents potential targets for metabolic engineering.

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

BackgroundMicroalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage.ResultsHere, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance.ConclusionsWe used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.

Sustainable production of photosynthetic isobutanol and 3-methyl-1-butanol in the cyanobacterium Synechocystis sp. PCC 6803

BackgroundCyanobacteria are emerging as green cell factories for sustainable biofuel and chemical production, due to their photosynthetic ability to use solar energy, carbon dioxide and water in a direct process. The model cyanobacterial strain Synechocystis sp. PCC 6803 has been engineered for the isobutanol and 3-methyl-1-butanol production by introducing a synthetic 2-keto acid pathway. However, the achieved productions still remained low. In the present study, diverse metabolic engineering strategies were implemented in Synechocystis sp. PCC 6803 for further enhanced photosynthetic isobutanol and 3-methyl-1-butanol production.ResultsLong-term cultivation was performed on two selected strains resulting in maximum cumulative isobutanol and 3-methyl-1-butanol titers of 1247 mg L−1 and 389 mg L−1, on day 58 and day 48, respectively. Novel Synechocystis strain integrated with a native 2-keto acid pathway was generated and showed a production of 98 mg isobutanol L−1 in short-term screening experiments. Enhanced isobutanol and 3-methyl-1-butanol production was observed when increasing the kivdS286T copy number from three to four. Isobutanol and 3-methyl-1-butanol production was effectively improved when overexpressing selected genes of the central carbon metabolism. Identified genes are potential metabolic engineering targets to further enhance productivity of pyruvate-derived bioproducts in cyanobacteria.ConclusionsEnhanced isobutanol and 3-methyl-1-butanol production was successfully achieved in Synechocystis sp. PCC 6803 strains through diverse metabolic engineering strategies. The maximum cumulative isobutanol and 3-methyl-1-butanol titers, 1247 mg L−1 and 389 mg L−1, respectively, represent the current highest value reported. The significantly enhanced isobutanol and 3-methyl-1-butanol production in this study further pave the way for an industrial application of photosynthetic cyanobacteria-based biofuel and chemical synthesis from CO2.

Transcriptional insights into Chlorella sp. ABC-001: a comparative study of carbon fixation and lipid synthesis under different CO2 conditions

BackgroundMicroalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content—an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases.ResultsCells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae.ConclusionsWe expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.

Construction of Glucose-6-Phosphate Dehydrogenase Overexpression Strain of Schizochytrium sp. H016 to Improve Docosahexaenoic Acid Production.

Docosahexaenoic acid (DHA) is an important omega-3 polyunsaturated fatty acid (PUFA) that plays a critical physiological role in human health. Schizochytrium sp. is considered an excellent strain for DHA production, but the synthesis of DHA is limited by the availability of nicotinamide adenine dinucleotide phosphate (NADPH). In this study, the endogenous glucose-6-phosphate dehydrogenase (G6PD) gene was overexpressed in Schizochytrium sp. H016. Results demonstrated that G6PD overexpression increased the availability of NADPH, which ultimately altered the fatty acid profile, resulting in a 1.91-fold increase in DHA yield (8.81 g/L) and increased carbon flux by shifting it from carbohydrate and protein synthesis to lipid production. Thus, G6PD played a vital role in primary metabolism. In addition, G6PD significantly increased DHA content and lipid accumulation by 31.47% and 40.29%, respectively. The fed-batch fermentation experiment results showed that DHA production reached 17.01 g/L in the overexpressing G6PD strain. These results elucidated the beneficial effects of NADPH on the synthesis of PUFA in Schizochytrium sp. H016, which may be a potential target for metabolic engineering. Furthermore, this study provides a promising regulatory strategy for the large-scale production of DHA in Schizochytrium sp.

SbMYB3 transcription factor promotes root-specific flavone biosynthesis in Scutellaria baicalensis.

Scutellaria baicalensis Georgi produces abundant root-specific flavones (RSFs), which provide various benefits to human health. We have elucidated the complete biosynthetic pathways of baicalein and wogonin. However, the transcriptional regulation of flavone biosynthesis in S. baicalensis remains unclear. We show that the SbMYB3 transcription factor functions as a transcriptional activator involved in the biosynthesis of RSFs in S. baicalensis. Yeast one-hybrid and transcriptional activation assays showed that SbMYB3 binds to the promoter of flavone synthase II-2 (SbFNSII-2) and enhances its transcription. In S. baicalensis hairy roots, RNAi of SbMYB3 reduced the accumulation of baicalin and wogonoside, and SbMYB3 knockout decreased the biosynthesis of baicalein, baicalin, wogonin, and wogonoside, whereas SbMYB3 overexpression enhanced the contents of baicalein, baicalin, wogonin, and wogonoside. Transcript profiling by qRT-PCR demonstrated that SbMYB3 activates SbFNSII-2 expression directly, thus leading to more abundant accumulation of RSFs. This study provides a potential target for metabolic engineering of RSFs.

PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila.

The key enzyme 6-phosphofructo-2-kinase (PFK2)/fructose-2,6-bisphosphatase (FBPase-2) is responsible for regulating the rates of glycolysis and gluconeogenesis in eukaryotes. However, its functions and mechanisms in filamentous fungi remain largely enigmatic. In this study, we systematically investigated the function of this enzyme in Myceliophthora thermophila, a thermophilic filamentous fungus with great capacity to produce industrial enzymes and organic acids. Our results showed that the M. thermophila genome encodes three isomers, all with the PFK2/FBPase-2 structure: pfk2-a, pfk2-b, and pfk2-c. Overexpression of each gene revealed that endogenous expression of pfk2-c (PFK2 activity) promoted glucose metabolism, while overexpression of pfk2-a (FBPase-2 activity) inhibited strain growth. Using knockouts, we found that each gene was individually non-essential, but the triple knockout led to significantly slower growth compared with the wild-type strain. Only the pfk2-a single knockout exhibited 22.15% faster sugar metabolism, exerted through activation of 6-phosphofructo-1-kinase (PFK1), thereby significantly promoting glycolysis and the tricarboxylic acid cycle. The FBPase-2 deletion mutant strain also exhibited overflow metabolism, and knocking out pfk2-a was proved to be able to improve the production and synthesis rate of various metabolites, such as glycerol and malate. This is the first study to systematically investigate the function of PFK2/FBPase-2 in a thermophilic fungus, providing an effective target for metabolic engineering in filamentous fungi.

Identification and functional characterization of three iridoid synthases in Gardenia jasminoides.

The discovery of three iridoid synthases (GjISY, GjISY2 and GjISY4) from Gardenia jasminoides and their functional characterization increase the understanding of iridoid scaffold/iridoid glycoside biosynthesis in iridoid-producing plants. Iridoids are a class of noncanonical monoterpenes that are found naturally in the plant kingdom mostly as glycosides. Over 40 iridoid glycosides (e.g., geniposide, gardenoside and shanzhiside) have been isolated from Gardenia jasminoides. They have multiple pharmacological properties and health-promoting effects. However, their biosynthetic pathway is poorly understood, and the iridoid synthase (ISY) responsible for the cyclization of the core scaffold remains unclear. In this study, three homologs of ISYs from G. jasminoides (GjISY, GjISY2 and GjISY4) were identified on the basis of transcriptomic data and functionally characterized. The genomic structure and intron-exon arrangement revealed that all three ISYs contained an intron. Biochemical assays indicated that all three recombinant enzymes reduced 8-oxogeranial to nepetalactol and its open forms (iridodials) as the products of the classical CrISY (Catharanthus roseus). In addition, all three enzymes reduced progesterone to 5-β-prognane-3,20-dione. However, only GjISY2 and GjISY4 reduced 2-cyclohexen-1-one to cyclohexanone. Overall, the GjISY2 expression levels in the flowers and fruits were similar to the GjISY and GjISY4 expression levels. By contrast, the GjISY2 expression levels in the upper and lower leaves were substantially higher than the GjISY and GjISY4 expression levels. Among the three, GjISY2 exhibited the highest catalytic efficiency for 8-oxogeranial. GjISY2 might be the major contributor to iridoid biosynthesis in G. jasminoides. Collectively, our results advance the understanding of iridoid scaffold/iridoid glycoside biosynthesis in G. jasminoides and provide a potential target for metabolic engineering and breeding.

Comparative physiological and transcriptomic profiles reveal regulatory mechanisms of soft rot disease resistance in Amorphophallus spp.

Amorphophallus spp. are important industrial crops in Southwest China. Unfortunately, bacterial disease is one of the factors limiting the development of the Amorphophallus spp. industry. Soft rot can be caused by the pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) and leads to large-scale losses in Amorphophallus spp. quality and production. However, the mechanisms underlying the response of Amorphophallus spp. to Pcc remain largely unknown. This study compared two species of Amorphophallus, A. muelleri and A. konjac, to explore Pcc resistance by combined physiological and transcriptome analysis. On the basis of phenotype analysis, A. muelleri exhibited higher resistance to Pcc than A. konjac. Moreover, the transcriptomes of A. muelleri and A. konjac were assembled, and differentially expressed genes (DEGs) were annotated. Significant categories including the plant hormone transduction pathway, phenylpropanoid biosynthesis pathway and plant pathogen interaction pathway were identified. Among these pathways, small auxin upregulated RNA (SAUR), ethylene responsive transcription factor 1 (ERF1), 4-coumarate-CoA ligase (4CL), peroxidase (POD), WRKY transcription factor, Ca2+ sensor-related and mitogen-activated protein kinase (MAPK) genes were upregulated in A. muelleri, which may be involved in the regulation of energy, oxidative stress and signal transduction upon exposure to Pcc infection. The transcription levels of four selected genes were further verified by quantitative real-time PCR. Overall, this study identifies several critical genes involved in resistance to soft rot disease in Amorphophallus spp. and provides a theoretical foundation for identifying potential targets for metabolic engineering for disease control.

Transcriptomic survey reveals multiple adaptation mechanisms in response to nitrogen deprivation in marine Porphyridium cruentum.

Single-cell red microalga Porphyridium cruentum is potentially considered to be the bioresource for biofuel and pharmaceutical production. Nitrogen is a kind of nutrient component for photosynthetic P. cruentum. Meanwhile, nitrogen stress could induce to accumulate some substances such as lipid and phycoerythrin and affect its growth and physiology. However, how marine microalga Porphyridium cruentum respond and adapt to nitrogen starvation remains elusive. Here, acclimation of the metabolic reprogramming to changes in the nutrient environment was studied by high-throughput mRNA sequencing in the unicellular red alga P. cruentum. Firstly, to reveal transcriptional regulation, de novo transcriptome was assembled and 8,244 unigenes were annotated based on different database. Secondly, under nitrogen deprivation, 2100 unigenes displayed differential expression (1134 upregulation and 966 downregulation, respectively) and some pathways including carbon/nitrogen metabolism, photosynthesis, and lipid metabolism would be reprogrammed in P. cruentum. The result demonstrated that nitrate assimilation (with related unigenes of 8–493 fold upregulation) would be strengthen and photosynthesis (with related unigenes of 6–35 fold downregulation) be impaired under nitrogen deprivation. Importantly, compared to other green algae, red microalga P. cruentum presented a different expression pattern of lipid metabolism in response to nitrogen stress. These observations will also provide novel insight for understanding adaption mechanisms and potential targets for metabolic engineering and synthetic biology in P. cruentum.

A WRKY transcription factor, PgWRKY4X, positively regulates ginsenoside biosynthesis by activating squalene epoxidase transcription in Panax ginseng

Ginsenosides are important metabolites synthesized by the traditional Chinese medicinal plant, Panax ginseng. The market demand for ginsenosides is increasing. Fungal elicitor is effective for improving ginsenosides biosynthesis. Here, we isolated an effective fungal elicitor, Chaetomium globosum from Panax notoginseng. Total saponins content in adventitious roots of P. ginseng was 3.94 times higher than that in control under C. globosum treatment. Correspondingly, the antioxidant activity of C. globosum-stressed roots is significantly increased than control adventitious roots and cultivated ginseng roots. In this study, PgWRKY4X, a novel pathogen-related gene encoding WRKY transcription factor from P. ginseng was isolated and functionally characterized. PgWRKY4X was responsive to the treatment of C. globosum. Subcellular localization assay indicated PgWRKY4X located in the nucleus. Electrophoretic mobility shift assay showed PgWRKY4X binds to the W-box of squalene epoxidase (PgSE) promoter. The interaction between PgWRKY4X and PgSE was discovered by glutathione S-transferase pull-down assay. Homology modeling and molecular docking was also performed to reveal the putative spatial interaction between PgWRKY4X and PgSE. Overexpression of PgWRKY4X in P. ginseng transgenic cells could significantly enhance ginsenosides accumulation by comprehensively upregulating ginsenosides biosynthetic genes, especially PgSE. Our study suggested that PgWRKY4X may be a potential target for metabolic engineering of ginsenosides biosynthesis in P. ginseng.

Application of metabolic network model to analyze intracellular metabolism of industrial microorganisms

To quickly and efficiently understand the intracellular metabolic characteristics of industrial microorganisms, and to find potential metabolic engineering targets, genome-scale metabolic network models (GSMMs) as a systems biology tool, are attracting more and more attention. We review here the 20-year history of metabolic network model, analyze the research status and development of GSMMs, summarize the methods for model construction and analysis, and emphasize the applications of metabolic network model for analyzing intracellular metabolic activity of microorganisms from cellular phenotypes, and metabolic engineering. Furthermore, we indicate future development trend of metabolic network model.

Glucose-6-Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis.

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

Microalgal assimilation of vitamin B12 toward the production of a superfood.

A network of components from different metabolic pathways is the building scaffold of an indispensable compound in the human organism-vitamin B12 . The biosynthesis of this compound is restricted to a limited number of representatives of bacteria and archaea, while vitamin B12 -dependent enzymes are spread through several domains of life. Different attempts have been performed to increase vitamin B12 levels in dietary products, particularly in vegetarian and vegan dietary regimes. The integration of vitamin B12 in microalgae through symbiosis with microorganisms generally recognized as safe, for example the probiotic Lactobacillus reuteri, can even increase the nutritional value of the microalgal biomass. This study reviews the microbial production of vitamin B12 based on genetic analyses and chemical studies. Recent genetic approaches are focused, particularly potential metabolic engineering targets to increase vitamin B12 production. The bioincorporation of vitamin B12 in microalgae as an attempt to provide a superfood is also reviewed. PRACTICAL APPLICATIONS: Novel food habits (i.e., vegan lifestyle) may lack relevant nutrients, including vitamin B12 . Therefore, there is an increased demand for dietary products rich in vitamin B12 . Of potential interest is the provision of microbial-based superfood rich in numerous nutrients, including this vitamin. This manuscript provides an in-depth and timely overview on vitamin B12 biosynthesis and the major advances on metabolic engineering for improved vitamin B12 production by probiotic bacteria and other microorganisms generally recognized as safe. A relevant advance would result from the bioincorporation of vitamin B12 in alternative microorganisms (non-vitamin B12 producers) increasingly recognized as superfood, that is microalgae.

Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism

BackgroundAcetate, an economical industrial chemical, which is also the precursor of acetyl-CoA, could serve as an alternative substrate for biomanufacturing. This nontraditional substrate can be widely produced from syngas via hydrolysis or pyrolysis of the cellulosic biomass, chemical or microbial catalysis, anaerobic fermentation in treated wastewater, etc. However, the toxicity of acetate to microorganisms has held back its utilization, especially for the eukaryotes that are good hosts for production of complicated pharmaceuticals or chemicals. This study seeks to improve acetate utilization in a widely used yeast host, Komagataella phaffii (previously Pichia pastoris), by metabolic engineering of acetate tolerance, transport, and metabolism.ResultsA kinase-deficient library of K. phaffii was firstly used to screen acetate-resistant kinases. The HRK1 knockout strain was sensitive to acetate and overexpression of this gene improved acetate tolerance and cell growth of the strain. Also, overexpression of HRK1 caused a 55% productivity improvement of acetyl-CoA-dependent 6-methylsalicylic acid (6-MSA). However, activation of Hrk1 on membrane H(+)-ATPase Pma1 seemed not to work in the engineered strain. Acetate transporter gene ScFPS1* was further overexpressed, despite of not improving 6-MSA biosynthesis. To enhance acetate metabolism, acetyl-CoA synthesizing related genes, yeast PpACS1, ScACS1*, and E. coli ackA/pta were overexpressed separately. Introduction of PpACS1 and ScACS1* each increased biosynthesis of 6-MSA by approximately 20% on 20 mM acetate. Finally, co-overexpression of HRK1 and ScACS1* improved 6-MSA productivity by 51% on 20 mM acetate, despite that a low expression level of HRK1 happened when genes were expressed under the same promoter.ConclusionsHRK1 screened by K. phaffii kinase-deficient library played an important role in acetate tolerance and was proved to profit the biosynthesis of acetyl-CoA-derived chemicals. It could be a potential target for metabolic engineering of acetate utilization in other eukaryotic hosts as well. A combined strategy of introducing genes for acetate tolerance and metabolism further improved biosynthesis of acetyl-CoA derived reporter compound in K. phaffii. This makes it a good choice for acetyl-CoA-derived chemicals with acetate as substrate or precursor in K. phaffii, which would also extend the use of this chassis host.

Comparative functional genomics of the acarbose producers reveals potential targets for metabolic engineering

The α-glucosidase inhibitor acarbose is produced in large-scale by strains derived from Actinoplanes sp. SE50 and used widely for the treatment of type-2 diabetes. Compared with the wild-type SE50, a high-yield derivative Actinoplanes sp. SE50/110 shows 2-fold and 3–7-fold improvement of acarbose yield and acb cluster transcription, respectively. The genome of SE50 was fully sequenced and compared with that of SE50/110, and 11 SNVs and 4 InDels, affecting 8 CDSs, were identified in SE50/110. The 8 CDSs were individually inactivated in SE50. Deletions of ACWT_4325 (encoding alcohol dehydrogenase) resulted in increases of acarbose yield by 25% from 1.87 to 2.34 g/L, acetyl-CoA concentration by 52.7%, and PEP concentration by 22.7%. Meanwhile, deletion of ACWT_7629 (encoding elongation factor G) caused improvements of acarbose yield by 36% from 1.87 to 2.54 g/L, transcription of acb cluster, and ppGpp concentration to 2.2 folds. Combined deletions of ACWT_4325 and ACWT_7629 resulted in further improvement of acarbose to 2.83 g/L (i.e. 76% of SE50/110), suggesting that the metabolic perturbation and improved transcription of acb cluster caused by these two mutations contribute substantially to the acarbose overproduction. Enforced application of similar strategies was performed to manipulate SE50/110, resulting in a further increase of acarbose titer from 3.73 to 4.21 g/L. Therefore, the comparative genomics approach combined with functional verification not only revealed the acarbose overproduction mechanisms, but also guided further engineering of its high-yield producers.

An enhanced genome-scale metabolic reconstruction of Streptomyces clavuligerus identifies novel strain improvement strategies.

In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

Reconstruction of genome-scale metabolic model of Yarrowia lipolytica and its application in overproduction of triacylglycerol

BackgroundYarrowia lipolytica is widely studied as a non-conventional model yeast owing to the high level of lipid accumulation. Therein, triacylglycerol (TAG) is a major component of liposome. In order to investigate the TAG biosynthesis mechanism at a systematic level, a novel genome-scale metabolic model of Y. lipolytica was reconstructed based on a previous model iYL619_PCP published by our lab and another model iYali4 published by Kerkhoven et al.ResultsThe novel model iYL_2.0 contains 645 genes, 1083 metabolites, and 1471 reactions, which was validated more effective on simulations of specific growth rate. The precision of 29 carbon sources utilities reached up to 96.6% when simulated by iYL_2.0. In minimal growth medium, 111 genes were identified as essential for cell growth, whereas 66 essential genes were identified in yeast extract medium, which were verified by database of essential genes, suggesting a better prediction ability of iYL_2.0 in comparison with other existing models. In addition, potential metabolic engineering targets of improving TAG production were predicted by three in silico methods developed in-house, and the effects of amino acids supplementation were investigated based on model iYL_2.0.ConclusionsThe reconstructed model iYL_2.0 is a powerful platform for efficiently optimizing the metabolism of TAG and systematically understanding the physiological mechanism of Y. lipolytica.