An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS''s.     

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 μM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 10⁶ spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.     

Isolation and characterization of a Klebsiella oxytoca strain for simultaneous azo-dye anaerobic reduction and bio-hydrogen production

A facultative anaerobic bacteria strain GS-4-08, isolated from an anaerobic sequence batch reactor for synthetic dye wastewater treatment, was investigated for azo-dye decolorization. This bacterium was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA gene analysis. It exhibited a good capacity of simultaneous decolorization and hydrogen production in the presence of electron donor. The hydrogen production was less affected even at a high Methyl Orange (MO) concentration of 0.5 mM, indicating a superior tolerability of this strain to MO. This efficient bio-hydrogen production from electron donor can not only avoid bacterial inhibition due to accumulation of volatile fatty acids during MO decolorization, but also can recover considerable energy from dye wastewater.     

Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans

Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.     

Anaerobic Spirochete from a Deep-Sea Hydrothermal Vent

An obligately anaerobic spirochete, designated strain GS-2, was selectively isolated from samples collected at a deep-sea (2,550 m) hydrothermal vent of the Galapagos Rift ocean floor spreading center. The morphological and physiological characteristics of strain GS-2 resembled those of Spirochaeta strains. However, strain GS-2 failed to grow consistently in any liquid medium tested. In addition, strain GS-2 grew more slowly and to lower yields than other Spirochaeta species. The occurrence of obligately anaerobic bacteria in hydrothermal vents indicates that the water in at least some of the vent areas is anoxic. The presence of strain GS-2 shows that these areas are favorable for anaerobic marine spirochetes.     

A novel platform for automated high-throughput fluxome profiling of metabolic variants

Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for ¹³C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources and screening capacity were achieved. This will undoubtedly facilitate the development of efficient cell factories.     

Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds

The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of aromatic heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial “chassis” for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative — to the previously constructed recombinant dsz cassettes — for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and once secreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient production of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures.     

Engineering cyanobacteria as cell factories for direct trehalose production from CO2

Trehalose is a non-reducing disaccharide with a wide range of applications in food, cosmetic, and pharmaceutical industries. Cyanobacteria are promising cell factories to produce biochemicals by using solar energy and CO₂. Trehalose is biosynthesized at low intracellular concentrations as a salt-inducible compatible solute in some cyanobacteria. In the current study, we demonstrated the efficient trehalose production without salt induction in cyanobacteria by metabolic engineering. The trehalose transporter 1 (TRET1) from an anhydrobiotic insect (Polypedilum vanderplanki) was successfully expressed in the engineered strains and the intracellular trehalose was efficiently secreted to the medium. As the results, the engineered strain co-expressing maltooligosyl trehalose synthase (MTS), maltooligosyl trehalose trehalohydrolase (MTH) and TRET1 secreted 97% of trehalose to the medium, and the titer was up to 2.7 g/L in 15 days. In addition, 5.7 g/L trehalose was produced by semi-continuous cultivation in 34 days. Taken together, this work demonstrates cyanobacteria can be applied as cell factories for direct sunlight-driven conversion of CO₂ into excreted trehalose.     

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.     

Identification of Genes Affecting Hydrogen Sulfide Formation in Saccharomyces cerevisiae

A screen of the Saccharomyces cerevisiae deletion strain set was performed to identify genes affecting hydrogen sulfide (H₂S) production. Mutants were screened using two assays: colony color on BiGGY agar, which detects the basal level of sulfite reductase activity, and production of H₂S in a synthetic juice medium using lead acetate detection of free sulfide in the headspace. A total of 88 mutants produced darker colony colors than the parental strain, and 4 produced colonies significantly lighter in color. There was no correlation between the appearance of a dark colony color on BiGGY agar and H₂S production in synthetic juice media. Sixteen null mutations were identified as leading to the production of increased levels of H₂S in synthetic juice using the headspace analysis assay. All 16 mutants also produced H₂S in actual juices. Five of these genes encode proteins involved in sulfur containing amino acid or precursor biosynthesis and are directly associated with the sulfate assimilation pathway. The remaining genes encode proteins involved in a variety of cellular activities, including cell membrane integrity, cell energy regulation and balance, or other metabolic functions. The levels of hydrogen sulfide production of each of the 16 strains varied in response to nutritional conditions. In most cases, creation of multiple deletions of the 16 mutations in the same strain did not lead to a further increase in H₂S production, instead often resulting in decreased levels.     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

Multidisciplinary Evidences that Synechocystis PCC6803 Exopolysaccharides Operate in Cell Sedimentation and Protection against Salt and Metal Stresses

Little is known about the production of exopolysaccharides (EPS) in cyanobacteria, and there are no genetic and physiological evidences that EPS are involved in cell protection against the frequently encountered environmental stresses caused by salt and metals. We studied four presumptive EPS production genes, sll0923, sll1581, slr1875 and sll5052, in the model cyanobacterium Synechocystis PCC6803, which produces copious amounts of EPS attached to cells (CPS) and released in the culture medium (RPS) as shown here. We show that sll0923, sll1581, slr1875 and sll5052 are all dispensable to the growth of all corresponding single and double deletion mutants in absence of stress. Furthermore, we report that sll0923, sll1581 and slr1875 unambiguously operate in the production of both CPS and RPS. Both sll1581 and slr1875 are more important than sll0923 for CPS production, whereas the contrary is true for RPS production. We show that the most EPS-depleted mutant, doubly deleted for sll1581 and slr1875, lacks the EPS mantle that surrounds WT cells and sorbs iron in their vicinity. Using this mutant, we demonstrate for the first time that cyanobacterial EPS directly operate in cell protection against NaCl, CoCl₂, CdSO₄ and Fe-starvation. We believe that our EPS-depleted mutants will be useful tools to investigate the role of EPS in cell-to-cell aggregation, biofilm formation, biomineralization and tolerance to environmental stresses. We also suggest using the fast sedimenting mutants as biotechnological cell factories to facilitate the otherwise expensive harvest of the producer cell biomass and/or its separation from products excreted in the growth media.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.     

Multi-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin

Biotin (Vitamin H or B₇) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.     

高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种

[背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。[目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。[方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。[结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。[结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。