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Fulltext Constitutive overexpression of Qui-Quine Starch gene simultaneously improves starch and protein content in bioengineered cassava (Manihot esculenta Crantz)

Hankoua BB, Diao M, Ligaba-Osena A, Garcia RA, Harun S, Ahlawat YK

Front Plant Sci, 2024;15:1442324.
PMID: 40066347 DOI: 10.3389/fpls.2024.1442324

Cassava is a crucial source of daily calorie intake for millions of people in sub-Saharan Africa (SSA) but has an inferior protein content. Despite numerous attempts utilizing both traditional and biotechnological methods, efforts to address protein deficiency in cassava have yet to meet with much success. We aim to leverage modern biotechnologies to enhance cassava's nutritional value by creating bioengineered cassava cultivars with increased protein and starch content. In this study, we utilized Qui-Quine Starch (QQS), a novel orphan gene unique to Arabidopsis thaliana, to develop transgenic cassava plants with increased protein and starch accumulation in their tissues. A total of 10 independent transgenic cassava lines expressing QQS were successfully regenerated in this study, among which line R7 (F) demonstrated superior growth vigor. Quantitative RT-PCR verified the expression of the QQS gene in the transgenic lines. Data showed that QQS expression in cassava plants increased leaf protein content by 36% in line R''' (LA) L2 and root protein by 17% for the same line compared to their wild-type and empty vector (NPTII) control plants. Moreover, leaf-soluble total carbohydrates increased by 51.76% in line R (G) L2, and root-soluble total carbohydrates increased by 46.75% in line R7 (F). The novel function of QQS in increasing the starch content in the transgenic biomass is demonstrated. No significant change in the content of specific amino acids was observed among the lines and various plant parts. In addition, QQS expression revealed increased biomass, plant vigor, and early In vitro mini-tubers production for line R7 (F). Gene interaction study between AtQQS and 59 interacting partners generated 184 interactions or edges. These gene networks comprised several functional categories regulating the starch metabolic and auxin biosynthetic processes. The role of QQS in imparting starch and protein content of transgenic cassava plants is validated. The next logical step is the evaluation of biochemical profiles of cassava lines expressing QQS that reach maturity and the transferability of these findings to consumer-preferred cassava cultivars and local landraces grown in SSA. This study represents the first biotechnological report demonstrating a simultaneous increase of protein and starch content in bioengineered cassava.
Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus

Ahlawat YK, Biswal AK, Harun S, Harman-Ware AE, Doeppke C, Sharma N, et al.

Biotechnol Biofuels Bioprod, 2024 Jan 13;17(1):5.
PMID: 38218877 DOI: 10.1186/s13068-023-02452-7

BACKGROUND: Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression.
RESULTS: We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants.
CONCLUSIONS: Overall, saccharification efficiency was increased by 35-50%, 21-42%, and 8-39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

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