From carbon dioxide to starch: no plants required: Many plants turn glucose from photosynthesis into polymers that form insoluble starch granules ideal for long-term energy storage in roots and seeds. Cai et al . developed a hybrid system in which carbon dioxide is reduced to methanol by an inorganic catalyst and then converted by enzymes first to three and six carbon sugar units and then to polymeric starch. This artificial starch anabolic pathway relies on engineered recombinant enzymes from many different source organisms and can be tuned to produce amylose or amylopectin at excellent rates and efficiencies relative to other synthetic carbon fixation systems—and, depending on the metric used, even to field crops. —MAF

Biomanufacturing is a type of manufacturing that utilizes biological systems (e.g., living microorganisms, resting cells, animal cells, plant cells, tissues, enzymes, or in vitro synthetic (enzymatic) systems) to produce commercially important biomolecules for use in the agricultural, food, material, energy, and pharmaceutical industries. History of biomanufacturing could be classified into the three revolutions in terms of respective product types (mainly), production platforms, and research technologies. Biomanufacturing 1.0 focuses on the production of primary metabolites (e.g., butanol, acetone, ethanol, citric acid) by using mono-culture fermentation; biomanufacturing 2.0 focuses on the production of secondary metabolites (e.g., penicillin, streptomycin) by using a dedicated mutant and aerobic submerged liquid fermentation; and biomanufacturing 3.0 focuses on the production of large-size biomolecules—proteins and enzymes (e.g., erythropoietin, insulin, growth hormone, amylase, DNA polymerase) by using recombinant DNA technology and advanced cell culture. Biomanufacturing 4.0 could focus on new products, for example, human tissues or cells made by regenerative medicine, artificial starch made by in vitro synthetic biosystems, isobutanol fermented by metabolic engineering, and synthetic biology-driven microorganisms, as well as exiting products produced by far better approaches. Biomanufacturing 4.0 would help address some of the most important challenges of humankind, such as food security, energy security and sustainability, water crisis, climate change, health issues, and conflict related to the energy, food, and water nexus.

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. 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. 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.

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 alkaliphilicIdiomarinasp. C9-1 was cloned and expressed inEscherichia 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. Ca²⁺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⁻¹and 99511.9 U mg⁻¹, theK_mvalues of 3.76 mg ml⁻¹and 3.98 mg ml⁻¹, and theV_maxvalues of 57538.5 U mg⁻¹and 108722.1 U mg⁻¹, respectively. Secreted expression of AprA-PPC inBacillus subtilisafter 48 h cultivation resulted in yield of 4935.5 U ml⁻¹with productivity of 102.8 U ml⁻¹h⁻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.