Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5′ proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes. Using the developed toolbox, we show that different 5′ proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.

Abbreviations:A1AT, alpha-1-antitrypsin; AATD, alpha-1-antitrypsin deficiency; AUC, area under curve; B3gnt2, UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2; Cas9, CRISPR-associated protein 9; C1INH, C1 esterase inhibitor; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeats; FACS, fluorescence-activated cell sorting; FITC, Fluorescein isothiocyanate; Fut8, alpha-(1,6)-fucosyltransferase; Glul, glutamate-ammonia ligase; HAE, hereditary angioedema; HM, high-mannose; indel, insertion or deletion; IVC, integral of viable cells; KO, knock-out; mAb, monoclonal antibody; Mgat4A, mannosyl (alpha-1,3-)-glycoprotein beta-1,4-Nacetylglucosaminyltransferase isozyme A; Mgat4B, mannosyl (alpha-1,3-)glycoprotein beta-1,4-N-acetylglucosaminyltransferase isozyme B; Mgat5, mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase; MSX, methionine sulfoximine; sgRNA, single guide RNA; SNA, sambucus nigra agglutinin; Sppl3, signal peptide peptidase like 3; St3gal3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; St3gal4, ST3 beta-galactoside alpha-2,3-sialyltransferase 4; St3gal6, ST3 beta-galactoside alpha-2,3-sialyltransferase 6; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; VCD, viable cell density; WT, wild type Abstract Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency.Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.