Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States of America.
Department of Neurobiology & Anatomy, University of Utah, Salt Lake City, Utah 84112, United States of America.
Biochim Biophys Acta Gen Subj. 2021 Jan;1865(1):129765. doi: 10.1016/j.bbagen.2020.129765. Epub 2020 Oct 16.
Heparin, a lifesaving blood thinner used in over 100 million surgical procedures worldwide annually, is currently isolated from over 700 million pigs and ~200 million cattle in slaughterhouses worldwide. Though animal-derived heparin has been in use over eight decades, it is a complex mixture that poses a risk for chemical adulteration, and its availability is highly vulnerable. Therefore, there is an urgent need in devising bioengineering approaches for the production of heparin polymers, especially low molecular weight heparin (LMWH), and thus, relying less on animal sources. One of the main challenges, however, is the rapid, cost-effective production of low molecular weight heparosan, a precursor of LMWH and size-defined heparosan oligosaccharides. Another challenge is N-sulfation of N-acetyl heparosan oligosaccharides efficiently, an essential modification required for subsequent enzymatic modifications, though chemical and enzymatic N-sulfation is effectively performed at the polymer level.
To devise a strategy to produce low molecular weight heparosan and heparosan oligosaccharides, several non-pathogenic E. coli strains are engineered by transforming the essential heparosan biosynthetic genes with or without the eliminase gene (elmA) from pathogenic E. coli K5.
The metabolically engineered non-pathogenic strains are shown to produce ~5 kDa heparosan, a precursor for low molecular weight heparin, for the first time. Additionally, heparosan oligosaccharides of specific sizes ranging from tetrasaccharide to dodecasaccharide are directly generated, in a single step, from the recombinant bacterial strains that carry both heparosan biosynthetic genes and the eliminase gene. Various modifications, such as chemical N-sulfation of N-acetyl heparosan hexasaccharide following the selective protection of reducing end GlcNAc residue, enzymatic C5-epimerization of N-sulfo heparosan tetrasaccharide and complete 6-O sulfation of N-sulfo heparosan hexasaccharide, are shown to be feasible.
We engineered non-pathogenic E. coli strains to produce low molecular weight heparosan and a range of size-specific heparosan oligosaccharides in a controlled manner through modulating culture conditions. We have also shown various chemical and enzymatic modifications of heparosan oligosaccharides.
Heparosan is a precursor of heparin and the methods to produce low molecular weight heparosan is widely awaited. The methods described herein are promising and will pave the way for potential large scale production of low molecular weight heparin anticoagulants and bioactive heparin oligosaccharides in the coming decade.
肝素是一种在全球每年超过 1 亿例手术中使用的救命抗凝剂，目前从全球屠宰场的 7 亿多只猪和 2 亿多头牛中提取。尽管动物源性肝素已经使用了八十多年，但它是一种复杂的混合物，存在化学掺假的风险，而且其供应非常脆弱。因此，迫切需要设计生产肝素聚合物的生物工程方法，特别是低分子量肝素（LMWH），从而减少对动物来源的依赖。然而，主要挑战之一是快速、经济高效地生产低分子量肝素聚糖，这是 LMWH 的前体和大小限定的肝素聚糖低聚糖。另一个挑战是有效进行 N-乙酰肝素聚糖低聚糖的 N-硫酸化，这是后续酶修饰所必需的修饰，尽管化学和酶 N-硫酸化在聚合物水平上有效进行。
为了设计生产低分子量肝素聚糖和肝素聚糖低聚糖的策略，通过转化致病性 E. coli K5 的必需肝素聚糖生物合成基因，以及或不转化消除酶基因（elmA），对几种非致病性 E. coli 菌株进行工程改造。
首次显示，代谢工程改造的非致病性菌株能够首次生产约 5 kDa 的肝素聚糖，这是低分子量肝素的前体。此外，直接从携带肝素聚糖生物合成基因和消除酶基因的重组细菌菌株中，一步生成特定大小的肝素聚糖低聚糖，范围从四糖到十二糖。已经显示各种修饰是可行的，例如 N-乙酰肝素六糖的选择性还原端 GlcNAc 残基保护下的化学 N-硫酸化、N-硫酸肝素四糖的 C5-差向异构化以及 N-硫酸肝素六糖的完全 6-O 硫酸化。
我们通过调节培养条件，设计了非致病性 E. coli 菌株，以可控的方式生产低分子量肝素聚糖和一系列特定大小的肝素聚糖低聚糖。我们还展示了肝素聚糖低聚糖的各种化学和酶修饰。
肝素聚糖是肝素的前体，生产低分子量肝素聚糖的方法备受期待。本文所述的方法很有前途，将为未来十年低分子量肝素抗凝剂和生物活性肝素聚糖低聚糖的潜在大规模生产铺平道路。
