Synthetic microbial communities have emerged as an attractive route for chemical bioprocessing. They are argued to be superior to single strains through microbial division of labor (DOL), but the exact mechanism by which DOL confers advantages remains unclear. Here, we utilize a synthetic Saccharomyces cerevisiae consortium along with mathematical modeling to achieve tunable mixed sugar fermentation to overcome the limitations of single-strain fermentation. The consortium involves two strains with each specializing in glucose or xylose utilization for ethanol production. By controlling initial community composition, DOL allows fine tuning of fermentation dynamics and product generation. By altering inoculation delay, DOL provides additional programmability to parallelly regulate fermentation characteristics and product yield. Mathematical models capture observed experimental findings and further offer guidance for subsequent fermentation optimization. This study demonstrates the functional potential of DOL in bioprocessing and provides insight into the rational design of engineered ecosystems for various applications.

Rewiring the metabolic flux for efficient microbial conversion requires robust, scalable gene assembly. However, conventional gene assembly approaches are labor-intensive, highly experience-dependent, and require extensive expertise to ensure reproducibility and efficiency. Even with advanced automation platforms such as biofoundries, assembling gene arrays with multiple transcriptional units (TUs) remains challenging. In this study, we present Efficient Modular Gene Assembly (EffiModular), an integrated in vitro and in vivo gene assembly platform compatible with automated workflows. EffiModular enables the assembly of up to eight TUs with 80% efficiency in a single transformation. Integrated into a biofoundry workflow, it enabled the construction of 120 distinct yeast strains with varying levels of expression of the β-carotene biosynthesis genes within 3 days. Compared with conventional approaches, it significantly reduces procedural complexity, minimizes reliance on operator expertise, and accelerates workflow timelines. These features establish EffiModular as a next-generation gene assembly platform for scalable, reproducible gene assembly in biofoundry-based genetic engineering.

A bstract: Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD ⁺ -linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two β-xylosidases, GH43-2 and GH43-7 , and a xylodextrin transporter, CDT-2 , from Neurospora crassa , yielding the engineered strain SR8A6S3-CDT ₂ -GH43 _2/7 . Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1 , that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT ₂ -GH43 _2/7 , produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. Highlights: Integration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; Intracellular fermentation of XOS, acetate and xylose improved ethanol production; Deletion of both sor1 Δ and gre3 Δ reduced xylitol production.

Here, we present a protocol for multigene pathway construction in Saccharomyces cerevisiae using a connector-enabled workflow that integrates Golden Gate Assembly with yeast homologous recombination. We provide procedures for assembling Level 1 transcriptional unit (TU) fragments using standardized connectors and directly co-transforming them into S. cerevisiae to form Level 2 multigene plasmids. Compared to conventional approaches, this protocol significantly reduces procedural complexity, minimizes dependence on operator expertise, and substantially accelerates workflow timelines. For complete details on the use and execution of this protocol, please refer to Seong et al.¹.