| Hyaluronan（hyaluronic acid, HA） is a high-molecular mass and highly anionic glycosaminoglycans（GAG） found in the extracellular matrix of all vertebrates and the capsule of some bacteria. HA participates in numerous physiological processes, the biological functions and applications of HA depend on its molecular mass. In particular, low-molecular-weight（LMW） HA oligosaccharides have unique biological activities including the stimulation of cell differentiation, angiogenesis and anti-tumor. Therefore HA oligosaccharides have broad applications in food, cosmetics and medicine. Because of the broad range of molecular weights（>3000 Da） and poor qualities of LMW HA degraded by physical and chemical methods, and the considerably high price of commercial hyaluronidase in enzymatic degradation, limiting the applications of these methods in large-scale production of HA oligosaccharides.This project focuses on solving the bottlenecks of the preparation of HAase and HA oligosaccharides by employing enzyme engineering, synthetic biology and metabolic engineering technologies: Constructing a high-efficiency HA biosynthesis pathway in food-grade Bacillus subtilis 168;Cloning and investigating the first identified leech HAase gene LHyal; Achieving the functional expression and rational regulation of LHyal in B. subtilis; Firstly proposing the microbial production mode for specific-molecular-weight HA oligosaccharides and HAase, which could also be used to produce other LMW polysaccharides. Inspiring by some synthetic biology methods in this project and facilitating the further work, two novel tools with high throughput for DNA combinatorial editing and assembly have been developed correspondingly. Major results achieved in this work are highlighted below.（1） Authors applied the random amplification of c DNA ends polymerase chain reaction（RACE-PCR） approach to identify the first HAase-encoding gene LHyal（accession number KJ026763, encoding 489 amino acids） of the second group HAase（EC 3.2.1.36） from leech. The phylogenetic tree analysis suggested that LHyal should be a potential endo-β-glucuronidase（family 79）. By combining protein engineering and high-density culture, authors achieved high-level production(8.42×105 U m L-1) in the yeast Pichia pastoris secretory expression system. Leech LHyal exhibited superior enzymatic properties and high substrate specificity, which cannot hydrolyse chitin, heparin or chondroitin sulphate. Furthermore, analysis of the hydrolytic process suggested that this novel enzyme adopts a nonprocessive endolytic mode to preferentially digest longer HA chains, yielding a narrow-spectrum of specific HA oligosaccharides with different incubation times and HA4 and HA6 end products.（2） Based on the successful construction of HA biosynthetic pathway in B. subtilis, all the native pathway genes for the biosynthesis of the HA precursors UDP-Glc UA and UDP-Glc NAc were systematically analyzed to identify the rate-limiting enzymes（tua D, gta B, glm U, glm M and glm S） for HA biosynthesis. By coexpressing these committed genes and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 to 3.16 g L-1 with a molecular weight range of 1.40×106-1.83×106 Da.（3） After N-terminal engineering, the leech hyaluronidase LHyal was firstly actively expressed B. subtilis 168. By combining a ribosome-binding site engineering strategy and high throughput screening, the expression level of LHyal was rationally regulated with 70-fold differences from 2.14×103 to 1.58×105 U m L-1. Against this background, the HA production increased significantly from 3.16 to 4.35 g L-1 with the MW decreased substantially from1.69×106 to 2.20×103 Da. The production of high-molecular-weight HA further increased from 3.16 to 5.96 g L-1 with a fed-batch fermentation, especially coexpressing LHyal(1.62×106 U m L-1) substantially increased HA production from 5.96 to 19.38 g L-1（6.62×103 Da）. To our knowledge, this is the highest reported HA titer produced by a microbial strain.（4） For high-efficiency directed evolution of HA synthase and combinatorial optimization of HA biosynthetic pathway（regulatory elements and enzymes） in further work, authors present a versatile evolution method termed ―rapidly efficient combinatorial oligonucleotides for directed evolution‖（RECODE） for rapidly introducing multiple combinatorial mutations to the target DNA by combined action of a thermostable high-fidelity DNA polymerase and a thermostable DNA Ligase in one reaction system with Two-step or One-step PCR. RECODE showed powerful capacity in combinatorial editing of a target DNA（bases substitution, insertion and deletion）. By applying this method, authors rapidly constructed a variant library of the rpo S promoters（with activity of 8-460%）, generated variants with significant differences on substrate specificities from the highly specific leech LHyal（with more than 30 mutant residues）. Based on RECODE, a novel approach of combinatorial engineering of regulatory elements and pathway enzymes was proposed to optimize the heme biosynthetic pathway by combinatorial evolution of promoter, RBS and pathway enzymes（target product with 20-fold increase）.（5） Based on a lot of DNA constructions in this work and inspired by assembly methods used in this study, authors report a novel, scarless and sequence-independent DNA Assembly method using Thermal Exonucleases and Ligase（DATEL） that allows the rapid, reliable, seamless construction of genetic circuits and biological pathways from multiple DNA fragments in one tube. Under the optimized conditions, DATEL allowed the rapid（1–2 h） one-step assembly of 2-10 DNA fragments with high accuracy（between 100% and 74%）. Experimental comparison showed DATEL enables to outperform Gibson isothermal assembly on the assembly efficiency and colonies correct. These results demonstrate that DATEL method can be used to rapidly construct various diverse libraries of enzymes or synthetic pathways for the high-throughput screening of variants with desirable phenotypes（20-fold production capacities of carotene）. |
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