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Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by 13 C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future. The microbial synthesis of carbon-containing compounds from single carbon precursors is desirable, yet designed pathways to achieve this goal overlap with host metabolism. Here the authors design a de novo metabolic pathway to assimilate formaldehyde into acetyl-CoA that does not overlap with known metabolic networks.

Lactic acid (LA) is an important organic acid with broad industrial applications. Considered as an environmentally friendly alternative to petroleum-based plastic with a wide range of applications, polylactic acid has generated a great deal of interest and therefore the demand for optically pure l- or d-lactic acid has increased accordingly. Microbial fermentation is the industrial route for LA production. LA bacteria and certain genetic engineering bacteria are widely used for LA production. Although some fungi, such as Saccharomyces cerevisiae, are not natural LA producers, they have recently received increased attention for LA production because of their acid tolerance. The main challenge for LA bioproduction is the high cost of substrates. The development of LA production from cost-effective biomasses is a potential solution to reduce the cost of LA production. This review examined and discussed recent progress in optically pure l-lactic acid and optically pure d-lactic acid fermentation. The utilization of inexpensive substrates is also focused on. Additionally, for PLA production, a complete biological process by one-step fermentation from renewable resources is also currently being developed by metabolically engineered bacteria. We also summarize the strategies and procedures for metabolically engineering microorganisms producing PLA. In addition, there exists some challenges to efficiently produce PLA, therefore strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are also discussed.

Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5’ untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis . Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms. To obtain optimal yield and productivity in bioproduction, expression of pathway genes must be appropriately coordinated. Here, the authors report repurposing of base editors for simultaneous regulation of multiple gene expression and demonstrate its application in industrially important and model microorganisms.

When dealing with computed tomography volume data, the accurate segmentation of lung nodules is of great importance to lung cancer analysis and diagnosis, being a vital part of computer-aided diagnosis systems. However, due to the variety of lung nodules and the similarity of visual characteristics for nodules and their surroundings, robust segmentation of nodules becomes a challenging problem. A segmentation algorithm based on the fast marching method is proposed that separates the image into regions with similar features, which are then merged by combining regions growing with k-means. An evaluation was performed with two distinct methods (objective and subjective) that were applied on two different datasets, containing simulation data generated for this study and real patient data, respectively. The objective experimental results show that the proposed technique can accurately segment nodules, especially in solid cases, given the mean Dice scores of 0.933 and 0.901 for round and irregular nodules. For non-solid and cavitary nodules the performance dropped—0.799 and 0.614 mean Dice scores, respectively. The proposed method was compared to active contour models and to two modern deep learning networks. It reached better overall accuracy than active contour models, having comparable results to DBResNet but lesser accuracy than 3D-UNet. The results show promise for the proposed method in computer-aided diagnosis applications.

Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here we report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. Visible light drives PSP efficiently into a long-lived triplet excited state (PSP*), which reacts rapidly with reduced nicotinamide adenine dinucleotide to generate a super-reducing radical (PSP•), which is strong enough to reduce many CO2-reducing catalysts. We determined the three-dimensional structure of PSP• at 1.8 Å resolution by X-ray crystallography. Genetic engineering enabled the site-specific attachment of a nickel–terpyridine complex and the modular optimization of the photochemical properties of PSP, the chromophore/catalytic centre distance and the catalytic centre microenvironment, which culminated in a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6%.

Background Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum . Results Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum . Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9 , allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum . This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869. Conclusions In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum . The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.

Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the “one enzyme-one substrate” model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94–96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols. Haloacid dehalogenase-like phosphatases are widespread across all domains of life and play a crucial role in the regulation of levels of sugar phosphate metabolites in cells. The authors report on the structure-guided engineering of phosphatases for dedicated substrate specificity for the conversion of sucrose and starch into fructose and mannose.

Alkaline proteases have a myriad of potential applications in many industrial processes such as detergent, food and feed production, waste management and the leather industry. In this study, we isolated several alkaline protease producing bacteria from soda lake soil samples. A novel serine alkaline protease (AprA) gene from alkaliphilic Idiomarina sp. C9-1 was cloned and expressed in Escherichia coli . The purified AprA and its pre-peptidase C-terminal (PPC) domain-truncated enzyme (AprA-PPC) showed maximum activity at pH 10.5 and 60 °C, and were active and stable in a wide range of pH and temperature. Ca 2+ significantly improved the thermostability and increased the optimal temperature to 70 °C. Furthermore, both AprA and AprA-PPC showed good tolerance to surfactants and oxidizing and reducing agents. We found that the PPC domain contributed to AprA activity, thermostability and surfactant tolerance. With casein as substrate, AprA and AprA-PPC showed the highest specific activity of 42567.1 U mg −1 and 99511.9 U mg −1 , the K m values of 3.76 mg ml −1 and 3.98 mg ml −1 , and the V max values of 57538.5 U mg −1 and 108722.1 U mg −1 , respectively. Secreted expression of AprA-PPC in Bacillus subtilis after 48 h cultivation resulted in yield of 4935.5 U ml −1 with productivity of 102.8 U ml −1 h −1 , which is the highest reported in literature to date. Without adding any lime or sodium sulfide, both of which are harmful pollutants, AprA-PPC was effective in dehairing cattle hide and skins of goat, pig and rabbit in 8–12 h without causing significant damage to hairs and grain surface. Our results suggest that AprA-PPC may have great potentials for ecofriendly dehairing of animal skins in the leather industry.

The flavonoid extract from Erigeron breviscapus , breviscapine, has increasingly been used to treat cardio- and cerebrovascular diseases in China for more than 30 years, and plant supply of E. breviscapus is becoming insufficient to satisfy the growing market demand. Here we report an alternative strategy for the supply of breviscapine by building a yeast cell factory using synthetic biology. We identify two key enzymes in the biosynthetic pathway (flavonoid-7- O -glucuronosyltransferase and flavone-6-hydroxylase) from E. breviscapus genome and engineer yeast to produce breviscapine from glucose. After metabolic engineering and optimization of fed-batch fermentation, scutellarin and apigenin-7- O -glucuronide, two major active ingredients of breviscapine, reach to 108 and 185 mg l –1 , respectively. Our study not only introduces an alternative source of these valuable compounds, but also provides an example of integrating genomics and synthetic biology knowledge for metabolic engineering of natural compounds. Breviscapine is the flavonoid extract from medical plant Erigeron breviscapus for the treatment of cardio- and cerebrovascular disease. Here, the authors identify the key enzymes of the biosynthetic pathway from the plant genome and engineer yeast to produce breviscapine from glucose.

Tricarboxylic acid cycle (TCA cycle) plays an important role for aerobic growth of heterotrophic bacteria. Theoretically, eliminating TCA cycle would decrease carbon dissipation and facilitate chemicals biosynthesis. Here, we construct an E. coli strain without a functional TCA cycle that can serve as a versatile chassis for chemicals biosynthesis. We first use adaptive laboratory evolution to recover aerobic growth in minimal medium of TCA cycle-deficient E. coli . Inactivation of succinate dehydrogenase is a key event in the evolutionary trajectory. Supply of succinyl-CoA is identified as the growth limiting factor. By replacing endogenous succinyl-CoA dependent enzymes, we obtain an optimized TCA cycle-deficient E. coli strain. As a proof of concept, the strain is engineered for high-yield production of four separate products. This work enhances our understanding of the role of the TCA cycle in E. coli metabolism and demonstrates the advantages of using TCA cycle-deficient E. coli strain for biotechnological applications. While tricarboxylic acid cycle (TCA cycle) is required for heterotrophic microbes, it reduces carbon yield of industrial products due to the release of excess CO2. Here, the authors construct an E. coli strain without a functional TCA cycle and demonstrate its feasibility as a chassis strain for production of four separate compounds.