Engineering marine fungi for conversion of d-galacturonic acid to mucic acid

Abstract

Background: 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. Results: 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 cosubstrate, 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. Conclusions: 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.

Use of ambr®250 to assess mucic acid production in fed-batch cultures of a marine Trichoderma sp. D-221704

“…Strain D-221704 (VTT culture collection, formerly referred to as T2) was engineered from Trichoderma sp LF328 (courtesy of Prof. Dr. Johannes Imhoff, GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany) by inserting a gene encoding uronate dehydrogenase under a strong synthetic promoter in the D-galacturonate reductase gene ( gar2 ), as described by Vidgren et al ( 2020b ). Spore suspensions were prepared by cultivating the fungus on potato-dextrose agar (BD, Sparks, Maryland, USA) for 7 days, after which the spores were collected from the surface of the culture in a solution containing 0.8% (w/v) NaCl, 0.025% (v/v) Tween20 and 20% (v/v) glycerol and stored at −80 °C.…”

“…It is available in relatively large quantities in the pectin found in citrus peel and sugar beet pulp, only part of which is needed or can be used in food products. Several publications have reported the use of genetically engineered fungi to convert D-galacturonic acid to mucic (galactaric) acid (Mojzita et al 2010 ; Kuivanen et al 2016 , 2019 ; Barth and Wiebe 2017 ; Vidgren et al 2020b ). Mucic acid is a symmetrical hexaric acid i.e.…”

“…Production in small scale batch cultures may be limited because of sequential use of the co-substrate and the D-galacturonic acid (Mojzita et al 2010 ) or by oxygen limitation. In fed-batch cultures, concentrations of 20 (Barth and Wiebe 2017 ; Paasikallio et al 2017 ) to 25 (Vidgren et al 2020b ) g L −1 have been obtained with the filamentous fungi Trichoderma reesei D-161646 and a marine Trichoderma sp. LF328 T2 (VTTCC D-221704).…”

“…The bioconversion process with D-161646 was successfully scaled up to 250 L, but the authors acknowledged that 20 g L −1 was still a low concentration for an efficient process, even though mucic acid can readily be precipitated from the solution at low pH (Paasikallio et al 2017 ). The recently developed T2 strain may have higher production potential than D-161646 (Vidgren et al 2020b ), but conditions for good production were not explored.…”

“…We describe here an assessment of pH, yeast extract and co-substrate concentrations in fed-batch cultivation to produce mucic acid from D-galacturonic acid using the marine Trichoderma sp. LF328 T2 (D-221704) that was genetically engineered to produce mucic acid by incorporating a uronate dehydrogenase gene under a strong synthetic promoter that would not be subject to glucose repression (Vidgren et al 2020b ). The cultivations were carried out in 110–150 mL cultures using an ambr ® 250 system, demonstrating its suitability for use with a filamentous fungus.…”