Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes

Abstract

Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae.

Increasing seed thiamin content impacts stored carbon partitioning and subsequent seedling stress tolerance

“…Indeed, 3-gene overexpressing lines contained higher levels of palmitic, stearic, and Oleic acids but lower levels of linolenic acid compared to the wild type seeds (Table 3). These results are consistent with the results reported by Higgins et al (2018) in which they showed that by the application of HMP and thiamin in the medium supplemented with glucose as organic substrate, algae cells reserved higher levels of palmitic and Oleic acids but the lower levels of linolenic acid.…”

Metabolic relationships of uncultured bacteria associated with the microalgae Gambierdiscus

“…However, they may convert HMP to TMP via thiamine‐phosphate pyrophosphorylase ( thiE ) and complete the phosphorylation step via thiamine‐monophosphate kinase ( thiL ) to form biologically active thiamine diphosphate (TDP). HMP produced by PHY‐1 and/or PHY‐2 may satisfy the hypothetical thiamine requirements of the Gambierdiscus host, although the ability to utilize this precursor varies by algal species and strain (McRose et al ., ; Paerl et al ., ; Higgins et al ., ; Higgins et al ., ). RHO‐5a contains the thiCDE genes but lacks thiL , suggesting it has the potential for de novo TMP synthesis but cannot form TDP.…”

Mechanisms of Sodium-Acetate-Induced DHA Accumulation in a DHA-Producing Microalga, Crypthecodinium sp. SUN

“…SUN. The pyruvate dehydrogenase component (PDH) catalyzed acetyl-CoA formation from the pyruvate formed during glycolysis [ 29 , 30 ]. Studies have shown that the upregulation of PDH was a key factor stimulating acetyl-CoA production and lipid accumulation in Chlamydomonas sp.…”