Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code.

Membrane proteins are critical to biological processes and central to life sciences and modern medicine. However, membrane proteins are notoriously challenging to study, mainly owing to difficulties dictated by their highly hydrophobic nature. Previously, we reported QTY code, which is a simple method for designing water-soluble membrane proteins. Here, we apply QTY code to a transmembrane receptor, histidine kinase CpxA, to render it completely water-soluble. The designed CpxAQTY exhibits expected biophysical properties and highly preserved native molecular function, including the activities of (i) autokinase, (ii) phosphotransferase, (iii) phosphatase, and (iv) signaling receptor, involving a water-solubilized transmembrane domain. We probe the principles underlying the balance of structural stability and activity in the water-solubilized transmembrane domain. Computational approaches suggest that an extensive and dynamic hydrogen-bond network introduced by QTY code and its flexibility may play an important role. Our successful functional preservation further substantiates the robustness and comprehensiveness of QTY code.

Metabolic engineering of Geobacillus thermoglucosidasius for polymer-grade lactic acid production at high temperature.

The production and application of biodegradable polylactic acid are still severely hindered by the cost of its polymer-grade lactic acid monomers. High-temperature biomanufacturing has emerged as an increasingly attractive approach to enable low-cost and high-efficiency bulk chemical production. In this study, thermophilic Geobacillus thermoglucosidasius was reprogrammed to obtain optically pure l-lactic acid- and d-lactic acid-producing strains, G. thermoglucosidasius GTD17 and GTD7, by using rational metabolic engineering strategies including pathway construction, by-product elimination, and production enhancing. Moreover, semi-rational adaptive evolution was carried out to further improve their lactic acid synthesis performance. The final strains GTD17-55 and GTD7-144 produce 151.1 g/L of l-lactic acid and 153.1 g/L of d-lactic acid at 60 °C, respectively. In consideration of the high temperature, productive performance of these strains is superior compared to the state-of-the-art industrial strains. This study lays the foundation for the low-cost and efficient production of biodegradable plastic polylactic acid.

Genomic Insights on the Carbon-Negative Workhorse: Systematical Comparative Genomic Analysis on 56 Synechococcus Strains.

Synechococcus, a type of ancient photosynthetic cyanobacteria, is crucial in modern carbon-negative synthetic biology due to its potential for producing bioenergy and high-value products. With its high biomass, fast growth rate, and established genetic manipulation tools, Synechococcus has become a research focus in recent years. Abundant germplasm resources have been accumulated from various habitats, including temperature and salinity conditions relevant to industrialization. In this study, a comprehensive analysis of complete genomes of the 56 Synechococcus strains currently available in public databases was performed, clarifying genetic relationships, the adaptability of Synechococcus to the environment, and its reflection at the genomic level. This was carried out via pan-genome analysis and a detailed comparison of the functional gene groups. The results revealed an open-genome pattern, with 275 core genes and variable genome sizes within these strains. The KEGG annotation and orthology composition comparisons unveiled that the cold and thermophile strains have 32 and 84 unique KO functional units in their shared core gene functional units, respectively. Each KO functional unit reflects unique gene families and pathways. In terms of salt tolerance and comparative genomics, there are 65 unique KO functional units in freshwater-adapted strains and 154 in strictly marine strains. By delving into these aspects, our understanding of the metabolic potential of Synechococcus was deepened, promoting the development and industrial application of cyanobacterial biotechnology.

Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase.

NADH-dependent d-lactate dehydrogenases (d-LDH) are important for the industrial production of d-lactic acid. Here, we identify and characterize an improved d-lactate dehydrogenase mutant (d-LDH1) that contains the Pro101Gln mutation. The specific enzyme activities of d-LDH1 toward pyruvate and NADH are 21.8- and 11.0-fold greater compared to the wild-type enzyme. We determined the crystal structure of Apo-d-LDH1 at 2.65 Å resolution. Based on our structural analysis and docking studies, we explain the differences in activity with an altered binding conformation of NADH in d-LDH1. The role of the conserved residue Pro101 in d-LDH was further probed in site-directed mutagenesis experiments. We introduced d-LDH1 into Bacillus licheniformis yielding a d-lactic acid production of 145.9 g L-1 within 60 h at 50°C, which was three times higher than that of the wild-type enzyme. The discovery of d-LDH1 will pave the way for the efficient production of d-lactic acid by thermophilic bacteria.

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis.

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways and overexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7 g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine.

The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3 g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility.

Microbial cell factories for bio-based biodegradable plastics production.

The misuse of petroleum-based plastics has resulted in serious environmental pollution and resource wastage. Biodegradable plastics can be used as green substitutes for traditional plastics. Here, we discuss the feasibility and technical bottlenecks in developing microbial cell factories for the production of biodegradable plastics from lignocellulosic wastes. First, we introduce the basic properties of the main biodegradable plastics on the market, including poly(lactic acid), poly(hydroxyalkanoate), and poly(butylene adipate-co-terephthalate). We then demonstrate the feasibility of synthesizing petroleum-based biodegradable plastic monomers from bio-based raw materials and propose strategies to further advance their commercial production through metabolic engineering and synthetic biology. We also analyze the main challenges facing the current development of bio-based biodegradable plastic biosynthesis technology. Finally, we discuss the current major lignocellulose bioconversion processes and explore way to further improve the utilization efficiency of the main carbohydrates in lignocellulosic hydrolysates by microorganisms, from the perspectives of sugar transport, sugar assimilation, and carbon catabolite inhibition.

Kinetic characteristics of long-term repeated fed-batch (LtRFb) l-lactic acid fermentation by a Bacillus coagulans strain.

Application of degradable plastics is the most critical solution to plastic pollution. As the precursor of biodegradable plastic PLA (polylactic acid), efficient production of l-lactic acid is vital for the commercial replacement of traditional plastics. Bacillus coagulans H-2, a robust strain, was investigated for effective production of l-lactic acid using long-term repeated fed-batch (LtRFb) fermentation. Kinetic characteristics of l-lactic acid fermentation were analyzed by two models, showing that cell-growth coupled production gradually replaces cell-maintenance coupled production during fermentation. With the LtRFb strategy, l-lactic acid was produced at a high final concentration of 192.7 g/L, on average, and a yield of up to 93.0% during 20 batches of repeated fermentation within 487.5 h. Thus, strain H-2 can be used in the industrial production of l-lactic acid with optimization based on kinetic modeling.

Steps Toward High-Performance PLA: Economical Production of d-Lactate Enabled by a Newly Isolated Sporolactobacillus terrae Strain.

Optically pure d-lactate production has received much attention for its critical role in high-performance polylactic acid production. However, the current technology can hardly meet the comprehensive demand of industrialization on final titer, productivity, optical purity, and raw material costs. Here, an efficient d-lactate producer strain, Sporolactobacillus terrae (S. terrae) HKM-1, is isolated for d-lactate production. The strain HKM-1 shows extremely high d-lactate fermentative capability by using peanut meal, soybean meal, or corn steep liquor powder as a sole nitrogen source; the final titers (205.7 g L-1 , 218.9 g L-1 , and 193.9 g L-1 , respectively) and productivities (5.56 g L-1 h-1 , 5.34 g L-1 h-1 , and 3.73 g L-1 h-1 , respectively) of d-lactate reached the highest level ever reported. A comparative genomic analysis between S. terrae HKM-1 and previously reported d-lactate high-producing Sporolactobacillus inulinus (S. inulinus) CASD is conducted. The results show that many unrelated genetic features may contribute to the superior performance in d-lactate production of S. terrae HKM-1. This d-lactate producer HKM-1, along with its fermentation process, is promising for sustainable d-lactate production by using agro-industrial wastes.