Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets.

Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and l-DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5-5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a l-DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Combinatorial engineering of betalain biosynthesis pathway in yeast Saccharomyces cerevisiae.

BACKGROUND: Betalains, comprising red-violet betacyanins and yellow-orange betaxanthins, are the hydrophilic vacuolar pigments that provide bright coloration to roots, fruits, and flowers of plants of the Caryophyllales order. Betanin extracted from red beets is permitted quantum satis as a natural red food colorant (E162). Due to antioxidant activity, betanin has potential health benefits. RESULTS: We applied combinatorial engineering to find the optimal combination of a dozen tyrosine hydroxylase (TyH) and 4,5-dopa-estradiol-dioxygenase (DOD) variants. The best-engineered Saccharomyces cerevisiae strains produced over six-fold higher betaxanthins than previously reported. By genome-resequencing of these strains, we found out that two copies of DOD enzyme from Bougainvillea glabra together with TyH enzymes from Abronia nealleyi, Acleisanthes obtusa, and Cleretum bellidiforme were present in the three high-betaxanthin-producing isolates. Next, we expressed four variants of glucosyltransferases from Beta vulgaris for betanin biosynthesis. The highest titer of betanin (30.8 ± 0.14 mg/L after 48 h from 20 g/L glucose) was obtained when completing the biosynthesis pathway with UGT73A36 glucosyltransferase from Beta vulgaris. Finally, we investigated betalain transport in CEN.PK and S288C strains of Saccharomyces cerevisiae and identified a possible role of transporter genes QDR2 and APL1 in betanin transport. CONCLUSIONS: This study shows the potential of combinatorial engineering of yeast cell factories for the biotechnological production of betanin.