Microbial lactic acid (LA) production under acidic fermentation conditions is favorable to reduce the production cost, but circumventing LA toxicity is a major challenge. A d-LA-producing Saccharomyces cerevisiae strain JHY5610 is generated by expressing d-lactate dehydrogenase gene (Lm. ldhA) from Leuconostoc mesenteroides, while deleting genes involved in ethanol production (ADH1, ADH2, ADH3, ADH4, and ADH5), glycerol production (GPD1 and GPD2), and degradation of d-LA (DLD1). Adaptive laboratory evolution of JHY5610 lead to a strain JHY5710 having higher LA tolerance and d-LA-production capability. Genome sequencing of JHY5710 reveal that SUR1 mutation increases LA tolerance and d-LA-production, whereas a loss-of-function mutation of ERF2 only contributes to increasing d-LA production. Introduction of both SUR1 and erf2Δ mutations into JHY5610 largely mimic the d-LA-production capability of JHY5710, suggesting that these two mutations, which could modulate sphingolipid production and protein palmitoylation, are mainly responsible for the improved d-LA production in JHY5710. JHY5710 is further improved by deleting PDC1 encoding pyruvate decarboxylase and additional integration of Lm. ldhA gene. The resulting strain JHY5730 produce up to 82.6 g L of d-LA with a yield of 0.83 g g glucose and a productivity of 1.50 g/(L · h) in fed-batch fermentation at pH 3.5.

The oleaginous yeast Yarrowia lipolytica has a tendency to use the non‐homologous end joining repair (NHEJ) over the homology directed recombination as double‐strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target‐AID (activation‐induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target‐specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target‐AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.

Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD+ regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose.

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Aims: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed‐batch cultures. Methods and Results: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3‐phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed‐batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5·3‐ to six‐fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. Conclusions: An effective conversion of xylulose to xylulose 5‐phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. Significance and Impact of the Study: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.

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A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.