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Microalgae are promising microorganisms used to produce value-added products or to develop sustainable approaches for environmental remediation. The ATP-binding cassette proteins (ABCs) of Chlamydomonas reinhardtii have been characterized as indispensable transporters for CO 2 concentrating mechanism, lipid biosynthesis, and heavy metal sequestration. However, few microalgal ABC proteins have been studied compared with higher plants or non-photosynthetic microorganisms. This study performed a genome-wide, evolutionary, and transcriptomic survey of C. reinhardtii ABC proteins (CrABCs). A total of 75 CrABCs were identified and classed into eight ABC subfamilies, from ABCA to ABCI. We found that no whole or partial genome duplication events occurred in C. reinhardtii after the ancient endosymbiosis events, but gene duplications occurred in a small range of chromosomal regions, which forced ABC family expansion. Abundant light, abscisic acid, and jasmonic acid response cis -elements were mapped in the CrABC promoters, coinciding with the evolutionary history of hormone signaling in Chlorophyta . The expression survey under light/dark rhythms revealed a close bond of CrABCs with cell division and development. A broad study of CrABCs supported their expected roles in heavy metal detoxification, lipid metabolism, and environmental adaptation. Moreover, the evolutionary and expression survey predicted the functions of unknown CrABCs, which are elaborated in the text. Two half-size CrABCGs-CrABCG3 and CrABCG26-were described as plasma-membrane transporters that might participate in lipidic compound secretion. This study provides fundamental and exhaustive information about CrABCs , which are indispensable for the functional elucidation of ABC proteins in microalgae., Competing Interests: The authors declare no conflicts of interest.

Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation and further clarifying the complex regulatory mechanism of lipid biosynthesis in the homeostasis of lipids, carbohydrates, and protein metabolism. Here, three lipid-related genes, DOF, LACS2, and CIS, were co-regulated in Chlamydomonas reinhartii by two circles of transformation to overexpress DOF and knock down LACS2 and CIS simultaneously. With the multiple regulations of these genes, the intracellular lipids and FA content increased by 142% and 52%, respectively, compared with CC849, whereas the starch and protein contents decreased by 45% and 24%. Transcriptomic analysis showed that genes in TAG and FA biosynthesis were up-regulated, and genes in starch and protein metabolism were down-regulated. This revealed that more carbon precursor fluxes from starch and protein metabolism were redirected towards lipid synthesis pathways. These results showed that regulating genes in various metabolisms contributed to carbon flux redirection and significantly improved intracellular lipids, demonstrating the potential of multiple gene regulation strategies and providing possible candidates for lipid overproduction in microalgae.

The use of genetic engineering techniques to alter the nutritional patterns of microalgae is a key means of enhancing microalgal biomass, as it enables the highly efficient heterotrophic growth of microalgae via the utilisation of organic carbon sources, especially glucose. To investigate the effects of the expression of the glucose transporter on the growth of the model alga, Chlamydomonas reinhardtii , we transformed C. reinhardtii using the human GLUT1 gene, regulated by the Hsp70 , PsaD , and Arg7 promoters, and obtained engineered algae after screening. The engineered algae showed better growth than the control algae under both heterotrophic and mixotrophic growth conditions. The engineered alga in which the GLUT1 gene was regulated by the Arg7 promoter exhibited the best growth. Although the engineered algae exhibited heterotrophic growth in complete darkness by using glucose as the carbon source, their growth rates and biomass were relatively low compared to those of the control. Analysis of the glucose transport ability of the engineered algae revealed that glucose was successfully transported to the cytoplasm via the glucose transporters, but was unable to enter the chloroplasts, which could explain the slow heterotrophic growth of the engineered algae. • Human glucose transporter GLUT1 was expressed in C. reinhardtii under the regulation of Hsp70 , PsaD , and Arg7 promoters. • Chlamydomonas can heterotrophic growth in the dark with glucose by expression of GLUT1. • Expression of Glut1 in C. reinharditii can successfully transport glucose to the cytoplasm, but not to the chloroplasts. [ABSTRACT FROM AUTHOR]