Two marine fungi, a Trichoderma sp. and a Coniochaeta sp., which can grow on d-galacturonic acid and pectin, were selected as hosts to engineer for mucic acid production, assessing the suitability of marine fungi for production of platform chemicals. The pathway for biotechnologcial production of mucic (galactaric) acid from d-galacturonic acid is simple and requires minimal modification of the genome, optimally one deletion and one insertion. d-Galacturonic acid, the main component of pectin, is a potential substrate for bioconversion, since pectin-rich waste is abundant. Trichoderma sp. LF328 and Coniochaeta sp. MF729 were engineered using CRISPR-Cas9 to oxidize d-galacturonic acid to mucic acid, disrupting the endogenous pathway for d-galacturonic acid catabolism when inserting a gene encoding bacterial uronate dehydrogenase. The uronate dehydrogenase was expressed under control of a synthetic expression system, which fucntioned in both marine strains. The marine Trichoderma transformants produced 25 g L−1 mucic acid from d-galacturonic acid in equimolar amounts: the yield was 1.0 to 1.1 g mucic acid [g d-galacturonic acid utilized]−1. d-Xylose and lactose were the preferred co-substrates. The engineered marine Trichoderma sp. was more productive than the best Trichoderma reesei strain (D-161646) described in the literature to date, that had been engineered to produce mucic acid. With marine Coniochaeta transformants, d-glucose was the preferred co-substrate, but the highest yield was 0.82 g g−1: a portion of d-galacturonic acid was still metabolized. Coniochaeta sp. transformants produced adequate pectinases to produce mucic acid from pectin, but Trichoderma sp. transformants did not. Both marine species were successfully engineered using CRISPR-Cas9 and the synthetic expression system was functional in both species. Although Coniochaeta sp. transformants produced mucic acid directly from pectin, the metabolism of d-galacturonic acid was not completely disrupted and mucic acid amounts were low. The d-galacturonic pathway was completely disrupted in the transformants of the marine Trichoderma sp., which produced more mucic acid than a previously constructed T. reesei mucic acid producing strain, when grown under similar conditions. This demonstrated that marine fungi may be useful as production organisms, not only for native enzymes or bioactive compounds, but also for other compounds.

中文翻译：

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工程海洋真菌将 D-半乳糖醛酸转化为粘酸。


两种海洋真菌，一种木霉菌。和一种可以在 d-半乳糖醛酸和果胶上生长的 Coniochaeta sp. 被选为宿主，进行粘酸生产工程，评估海洋真菌生产平台化学品的适用性。从 d-半乳糖醛酸生物技术生产粘液（半乳糖二）酸的途径很简单，并且需要对基因组进行最小程度的修改，最好是一次删除和一次插入。 d-半乳糖醛酸是果胶的主要成分，是生物转化的潜在底物，因为富含果胶的废物非常丰富。木霉属 sp. LF328 和 Coniochaeta sp。 MF729 使用 CRISPR-Cas9 进行工程改造，将 d-半乳糖醛酸氧化为粘酸，当插入编码细菌糖醛酸脱氢酶的基因时，会破坏 d-半乳糖醛酸分解代谢的内源途径。糖醛酸脱氢酶在合成表达系统的控制下表达，该系统在两种海洋菌株中都有功能。海洋木霉转化体从等摩尔量的 d-半乳糖醛酸产生 25 g L−1 粘酸：产量为 1.0 至 1.1 g 粘酸 [使用的 g d-半乳糖醛酸]−1。 d-木糖和乳糖是优选的辅助底物。工程海洋木霉属 sp。比迄今为止文献中描述的最好的里氏木霉菌株（D-161646）生产力更高，该菌株经过改造可产生粘酸。对于海洋Coniochaeta转化体，d-葡萄糖是首选的共底物，但最高产量为0.82 gg−1：一部分d-半乳糖醛酸仍然被代谢。 Coniochaeta sp。转化体产生足够的果胶酶以从果胶产生粘酸，但木霉属物种。转化体没有。 这两个海洋物种均使用 CRISPR-Cas9 成功改造，并且合成表达系统在这两个物种中均发挥作用。虽然Coniochaeta sp。转化子直接从果胶产生粘酸，D-半乳糖醛酸的代谢没有完全被破坏，粘酸含量低。海洋木霉转化体中的 d-半乳糖醛酸途径被完全破坏，在相似条件下生长时，该转化体比先前构建的里氏木霉产生粘酸的菌株产生更多的粘酸。这表明海洋真菌可用作生产生物体，不仅可以生产天然酶或生物活性化合物，还可以生产其他化合物。