Metabolic Engineering Communications最新文献

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

Terpenoids are a wide class of organic compounds with industrial relevance. The natural ability of cyanobacteria to produce terpenoids via the methylerythritol 4-phosphate (MEP) pathway makes these organisms appealing candidates for the generation of light-driven cell factories for green chemistry. Here we address the improvement of the production of (E)-α-bisabolene, a valuable biofuel feedstock, in Synechocystis sp. PCC 6803 via sequential heterologous expression of bottleneck enzymes of the native pathway. Expression of the bisabolene synthase is sufficient to complete the biosynthetic pathway of bisabolene. Expression of a farnesyl-pyrophosphate synthase from Escherichia coli did not influence production of bisabolene, while enhancement of the MEP pathway via additional overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and IPP/DMAPP isomerase (IDI) significantly increased production per cell. However, in the absence of a carbon sink, the overexpression of DXS and IDI leads to significant growth impairment. The final engineered strain reached a volumetric titre of 9 ​mg ​L⁻¹ culture of bisabolene after growing for 12 days. When the cultures were grown in a high cell density (HCD) system, we observed an increase in the volumetric titres by one order of magnitude for all producing-strains. The strain with improved MEP pathway presented an increase twice as much as the remaining engineered strains, yielding more than 180 ​mg ​L⁻¹ culture after 10 days of cultivation. Furthermore, the overexpression of these two MEP enzymes prevented the previously reported decrease in the bisabolene specific titres when grown in HCD conditions, where primary metabolism is usually favoured. We conclude that fine-tuning of the cyanobacterial terpenoid pathway is crucial for the generation of microbial platforms for terpenoid production on industrial-scale.

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23–55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.

This study employs biomass growth analyses and ¹³C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a β-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best β-carotene titer (121 ± 13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. In return, acetone produced by C. beijerinckii G117 was reduced into isopropanol by B. subtilis BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered B. subtilis. In the symbiotic system consisting of two strains, 1.7 ​g/L of acetone, 4.8 ​g/L of butanol, and 0.9 ​g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 ​g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 ​g/L of acetone, 4.9 ​g/L of butanol, and 0.2 ​g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 ​g/L when we increased the inoculum size of B. subtilis BsADH2 and optimized other process parameters. With the Bacillus-Clostridium co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of Clostridium. This strategy of employing a recombinant Bacillus to co-culture with Clostridium should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.

Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 ​mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.

Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 ​g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 ​g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 ​g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.

Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5–10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.

Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD, hag, and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD, hag, and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h^-1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.